Overexpression Of Thioredoxin Research Articles

Thioredoxin overexpression in HT-1080 cells induced cellular senescence and sensitization to gamma radiation

An increment of thioredoxin-1 (TRX) is observed in many human primary cancers and appears to contribute to an increase of cell growth and a resistance to chemotherapy. On the contrary, when TRX was overexpressed in the HT-1080 fibrosarcoma cells, the cell growth was retarded and chromosomal polyploidy and cellular senescence were induced. TRX-overexpression made HT-1080 cells resistant to an oxidative stress caused by H 2O 2 or paraquat. But these cells were significantly sensitive to ionizing radiation, showing an abrogation of the G 2 checkpoint. Their DNA contents were twice of the controls and they expressed typical senescence markers. Their expression levels of p53 and cyclin-dependent kinase inhibitors (CDKI) were about 2–3-fold higher than the control. Nevertheless, cyclin D1 and D3, which are negatively regulated by CDKIs, were also increased. Overall, in HT-1080 cells the TRX-overexpression created a state of cellular senescence caused by a simultaneous stimulation of the mitogen-activated pathways and an inhibition of the cyclin-dependent kinases, which is known as a hypermitogenic arrest.

Nitric oxide-induced persistent inhibition and nitrosylation of active site cysteine residues of mitochondrial cytochrome-c oxidase in lung endothelial cells

Persistent inhibition of cytochrome-c oxidase, a terminal enzyme of the mitochondrial electron transport chain, by excessive nitric oxide (NO) derived from inflammation, polluted air, and tobacco smoke contributes to enhanced oxidant production and programmed cell death or apoptosis of lung cells. We sought to determine whether the long-term exposure of pulmonary artery endothelial cells (PAEC) to pathophysiological concentrations of NO causes persistent inhibition of complex IV through redox modification of its key cysteine residues located in a putative NO-sensitive motif. Prolonged exposure of porcine PAEC to 1 mM 2,2'-(hydroxynitrosohydrazino)-bis-ethanamine (NOC-18; slow-releasing NO donor, equivalent to 1-5 microM NO) resulted in a gradual, persistent inhibition of complex IV concomitant with a reduction in ratios of mitochondrial GSH and GSSG. Overexpression of thioredoxin in mitochondria of PAEC attenuated NO-induced loss of complex IV activities, suggesting redox regulation of complex IV activity. Sequence analysis of complex IV subunits revealed a novel putative NO-sensitive motif in subunit II (S2). There are only two cysteine residues in porcine complex IV S2, located in the putative motif. Immunoprecipitation and Western blot analysis and "biotin switch" assay demonstrated that exposure of PAEC to 1 mM NOC-18 increased S-nitrosylation of complex IV S2 by 200%. Site-directed mutagenesis of these two cysteines of complex IV S2 attenuated NO-increased nitrosylation of complex IV S2. These results demonstrate for the first time that NO nitrosylates active site cysteines of complex IV, which is associated with persistent inhibition of complex IV. NO inhibition of complex IV via nitrosylation of NO-sensitive cysteine residues can be a novel upstream event in NO-complex IV signaling for NO toxicity in lung endothelial cells.

Overexpression of thioredoxin prevents thioacetamide-induced hepatic fibrosis in mice

Thioredoxin is a small redox-active protein with anti-oxidant and anti-apoptotic effects. We have previously reported that thioacetamide-induced acute hepatitis was attenuated in thioredoxin transgenic mice. The aim of the present study was to investigate the protective effect of thioredoxin for hepatic fibrosis. We subjected thioredoxin transgenic mice to thioacetamide-induced hepatic fibrosis. We also studied the effect of thioredoxin on the activation process of primary-cultured hepatic stellate cell. The expression of endogenous thioredoxin was induced in hepatocytes of thioacetamide-induced murine and rat fibrotic livers. Overexpression of thioredoxin inhibited tumor necrosis factor-alpha-induced apoptosis of HepG2 cells. Thioacetamide-induced fibrosis and accumulation of malondialdehyde were suppressed in transgenic mice as compared with wild type mice. Hepatic stellate cells isolated from transgenic mice were less proliferative than those isolated from wild type mice. Recombinant thioredoxin significantly inhibited DNA synthesis of primary-cultured stellate cells under serum or platelet-derived growth factor stimulation. Thioredoxin has a potential to attenuate hepatic fibrosis via suppressing oxidative stress and inhibiting proliferation of stellate cells.

Oxidative stress and thioredoxin-interacting protein promote intravasation of melanoma cells

Although intravasation may be a critical rate-limiting step in the metastatic cascade, the role of oxidative stress in intravasation is unknown. We tested the hypothesis that reactive oxygen species (ROS), regulated by thioredoxin interacting protein (Txnip) through the action of thioredoxin (Trx), influence human SK-MEL-28 melanoma cell reverse (basolateral-to-apical) transendothelial migration (TEM) in vitro as a model for intravasation. Reverse transendothelial migration was dose-dependently induced by hydrogen peroxide 2.4-fold for 0.1 μM ( P < 0.01) and 3.9-fold for 1 μM ( P < 0.001) vs. control, and this effect was blocked by the antioxidant N-acetylcysteine. Overexpression of Txnip by infecting melanoma cells with adenovirus increased TEM 3-fold vs. control ( P < 0.001), and this increase was blocked by N-acetylcysteine, indicating a redox-sensitive mechanism. Conversely, thioredoxin overexpression blocked hydrogen peroxide-induced TEM. Exposure to ultraviolet-A radiation increased ROS 1.8-fold ( P < 0.01), and this was accompanied by a 45% reduction ( P < 0.05) in thioredoxin activity and an 11.4-fold ( P < 0.001) increase in Txnip gene expression. These data suggest that TEM of melanoma cells during intravasation is in part mediated by ROS-sensitive cellular signaling cascades, may be controlled by Txnip and its interaction with thioredoxin that in turn modulates cellular levels of oxidative stress, and may be initiated by ultraviolet-A induction of this cascade.

Thioredoxin-interacting protein controls cardiac hypertrophy through regulation of thioredoxin activity.

Although cellular redox balance plays an important role in mechanically induced cardiac hypertrophy, the mechanisms of regulation are incompletely defined. Because thioredoxin is a major intracellular antioxidant and can also regulate redox-dependent transcription, we explored the role of thioredoxin activity in mechanically overloaded cardiomyocytes in vitro and in vivo. Overexpression of thioredoxin induced protein synthesis in cardiomyocytes (127+/-5% of controls, P<0.01). Overexpression of thioredoxin-interacting protein (Txnip), an endogenous thioredoxin inhibitor, reduced protein synthesis in response to mechanical strain (89+/-5% reduction, P<0.01), phenylephrine (80+/-3% reduction, P<0.01), or angiotensin II (80+/-4% reduction, P<0.01). In vivo, myocardial thioredoxin activity increased 3.5-fold compared with sham controls after transverse aortic constriction (P<0.01). Aortic constriction did not change thioredoxin expression but reduced Txnip expression by 40% (P<0.05). Gene transfer studies showed that cells that overexpress Txnip develop less hypertrophy after aortic constriction than control cells in the same animals (28.1+/-5.2% reduction versus noninfected cells, P<0.01). Thus, even though thioredoxin is an antioxidant, activation of thioredoxin participates in the development of pressure-overload cardiac hypertrophy, demonstrating the dual function of thioredoxin as both an antioxidant and a signaling protein. These results also support the emerging concept that the thioredoxin inhibitor Txnip is a critical regulator of biomechanical signaling.

Redox regulation by thioredoxin in cardiovascular diseases.

Increasing evidence has indicated that the modulation of intracellular redox states has important aspects to cellular events, such as cellular proliferation, activation, growth inhibition, or death via the regulation of intracellular signal transduction and gene expression. Thioredoxin (TRX) is a multifunctional stress-inducible protein, which protects cells from various types of stresses. TRX has not only a scavenging activity of reactive oxygen species, but also a regulating activity of various intracellular molecules including transcription factors. We demonstrated that the serum TRX levels are correlated with the severity of heart failure, and are negatively correlated with left ventricular ejection fractions of patients with heart failure. The expression of TRX is enhanced in endothelial cells and macrophages in human atherosclerotic plaques, in balloon-injured rat arteries, and in damaged cardiomyocytes of rats with acute myocarditis. Overexpression of TRX in transgenic mice attenuates adriamycin-induced cardiotoxicity by reducing oxidative stresses. These findings suggest that TRX and the redox system modulated by TRX have an important role in cellular defense against oxidative stress in cardiovascular diseases.

Overexpression of thioredoxin prevents acute hepatitis caused by thioacetamide or lipopolysaccharide in mice

Thioredoxin (Trx) is a small redox-active protein with antioxidant and antiapoptotic effects. Trx transgenic (Tg) mice are more resistant to cerebral infarction and survive longer than wild-type (WT) C57BL/6 mice. The aim of the present study was to investigate the protective role of Trx in acute hepatitis models. The expression of endogenous Trx was decreased in thioacetamide (TAA)-induced acute hepatitis. TAA (100 μg/g) was injected intraperitoneally in WT and Tg mice. Survival rate after TAA injection was higher in Tg mice than in WT mice. The level of oxidative stress was significantly less in Tg mice than in WT mice, as shown by the protein carbonylation assay and lipid peroxidation assay. Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL)-positive cells were less in Tg mice than in WT mice, which was consistent with DNA laddering assay. Caspase-3 and caspase-9 activities and cytochrome c release were significantly inhibited in Tg mice compared with those in WT mice. In addition, lipopolysaccharide (LPS) plus d-galactosamine (GalN), or anti- Fas antibody (Jo2) were injected. Survival rate after LPS plus GalN injection was much higher in Tg mice than in WT mice. In contrast, there was no difference in survival rate after Jo2 injection between WT and Tg mice. In conclusion, transgene of Trx attenuated TAA- or LPS-induced acute lethal hepatitis. In addition to an antioxidant effect, Trx has the potential to protect acute liver injury via an antiapoptotic effect, which mainly inhibits mitochondria-mediated apoptosis signaling. (H epatology 2003;37:1015-1025.)

Overexpression of thioredoxin-1 in transgenic mice prevents Helicobacter felis-induced gastritis

Overexpression of thioredoxin-1 in transgenic mice prevents Helicobacter felis-induced gastritis

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.

Overexpression of thioredoxin-1 in transgenic mice attenuates adriamycin-induced cardiotoxicity.

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. 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. TRX1 is upregulated by intracellular oxidative stress generated by ADR. TRX1 has a protective role against ADR-induced cardiotoxicity by reducing oxidative stress.

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.

Vitamin D3-up-regulated Protein-1 Is a Stress-responsive Gene That Regulates Cardiomyocyte Viability through Interaction with Thioredoxin

The protein-disulfide reductase thioredoxin is critical for redox signaling during apoptosis and growth. In this study, we demonstrate that vitamin D(3)-up-regulated protein-1 regulates thioredoxin in conditions of biomechanical or oxidative stress and critically regulates cardiomyocyte viability. Expression of vitamin D(3)-up-regulated protein-1 but not of thioredoxin in rat cardiomyocytes was rapidly suppressed by biomechanical strain or hydrogen peroxide at both mRNA and protein levels. Mechanical suppression of vitamin D(3)-up-regulated protein-1 gene expression was blocked by N-acetylcysteine. The half-life of vitamin D(3)-up-regulated protein-1 transcripts in cardiomyocytes was only 1.1 h and remained unchanged after mechanical stimulation, suggesting that rapid responses in vitamin D(3)-up-regulated protein-1 gene expression occur through transcriptional control. Vitamin D(3)-up-regulated protein-1 down-regulation by strain or hydrogen peroxide led to increased thioredoxin activity, whereas adenovirus-mediated overexpression of vitamin D(3)-up-regulated protein-1 suppressed thioredoxin activity. Overexpression of vitamin D(3)-up-regulated protein-1 but not of thioredoxin induced cardiomyocyte apoptosis. Furthermore, overexpression of vitamin D(3)-up-regulated protein-1 sensitized cells to hydrogen peroxide-induced apoptosis, whereas overexpression of thioredoxin protected against injury. These data identify vitamin D(3)-up-regulated protein-1 as a key stress-responsive inhibitory switch of thioredoxin activity in cardiomyocytes and demonstrate that the vitamin D(3)-up-regulated protein-1/thioredoxin axis has an important role in the preservation of cellular viability.

Attenuation of retinal photooxidative damage in thioredoxin transgenic mice

Thioredoxin (TRX) is an endogenous redox (reduction/oxidation) regulator that has cytoprotective effects against various types of oxidative stresses. Exposure to excessive levels of white light induces retinal photoreceptor damage. To test the cytoprotective effect of overexpressed TRX against retinal photooxidative damage, both TRX transgenic (trx-tg) mice and C57BL/6 (wild type) mice were exposed to intense white fluorescent light. The amounts of oxidized and tyrosine-phosphorylated proteins decreased in the neural retinas of the trx-tg mice compared to the wild type mice after light exposure. The electroretinographic amplitudes were higher and the formation of oxidized DNA was lower in trx-tg mice compared to wild type mice after light exposure. These results suggest that overexpression of TRX suppresses retinal photooxidative damage. TRX intensification may be a useful therapeutic strategy to prevent retinal photic injury.

Overexpressed thioredoxin compensates Fanconi anemia related chromosomal instability.

The cause of the molecular defect of Fanconi anemia (FA) remains unknown. Cells from patients with FA exert an elevated spontaneous chromosomal instability which is further triggered by mitomycin C. The induced lability is reduced by overexpression of thioredoxin which is not the case for spontaneous instability. However, both are eliminated by overexpression of thioredoxin cDNA with an added nuclear localization signal. This implies that thioredoxin is lacking in the nuclei of FA cells. The total thioredoxin content in all FA cells tested is reduced. The resultant lack of nuclear thioredoxin can be the explanation for the major symptomatology in FA. Since thioredoxin is known to be the reactive cofactor of ribonucleotid reductase its shortcoming reduces the supply of deoxyribonucleotides thus hindering the DNA and replication repair with resultant chromosomal breaks. Furthermore, depression of tyrosine hydroxylase, the key enzyme of melanine synthesis, could be the basis for the pathognomotic 'café au lait' spots of FA. The observation of thioredoxin reduction in FA cells permits insight into the molecular phathophysiology of FA.

Enhanced resistancy of thioredoxin-transgenic mice against influenza virus-induced pneumonia

Thioredoxin (TRX) is a small redox-active protein with anti-oxidant effect and redox-regulating functions. Using TRX transgenic (Tg) mice in which human TRX is overexpressed systemically under the control of β-actin promoter, the effects of influenza virus infection were examined in TRX Tg mice and wild type C57BL/6 mice. (1) Median lethal dose (LD 50) against influenza virus infection in wild-type C57BL/6 mice was 10 −5.3 dilution, while that of TRX Tg mice was 10 −4.2 dilution. Thus, TRX Tg mice were more resistant against the virus infection than wild-type mice. (2) The body weights of wild-type mice 7 days after infection with a sublethal dose of the virus (10 −6 dilution) decreased significantly, whereas those of TRX Tg mice increased slightly. (3) Histopathology of the lung at 3 weeks after sublethal infection of influenza virus showed that severe alveolar or bronchiolar destruction was observed in wild-type mice, while mild viral pneumonia was seen in the TRX Tg mice. (4) Local (IgA) and systemic (IgG) antibody productions against influenza virus hemagglutinin in mice surviving 3 weeks after infection were similar between wild-type and TRX Tg mice. These results indicate that overexpression of TRX in Tg mice suppresses the inflammatory overshoot of viral pneumonia caused by influenza virus infection, resulting in the reduction of mortality without affecting the host's systemic immune responses to the infection. TRX may play some important roles in regulating the inflammatory process in the primary host defense against infection.

Redox-modulated xenobiotic action and ROS formation: a mirror or a window?

A number of xenobiotics require redox reactions to form the reactive intermediates involved in the ultimate toxic events (e.g., adduct formation). The same mechanisms lead to the formation of reactive oxygen species (ROS), which can themselves exert direct toxicity including, e.g., DNA oxidative damage or glutathione depletion. The occurence of both mechanistic features in xenobiotic activation and toxicity may raise some difficulties in ascertaining the respective roles of reactive intermediates versus ROS-related mechnisms. An example is provided by the toxicity mechanisms of mitomycin C (MMC) and diepoxybutane (DEB), which are commonly referred to as 'cross-linkers'. Their toxic actions, however, are well-known to be modulated via redox parameters, such as oxygen tension, antioxidants levels, or thioredoxin overexpression. The diagnostic assessment of Fanconi's anaemia (FA) relies on MMC and DEB sensitivity, which is usually referred to as 'cross-linker sensitivity'; thus the redox-dependent toxicities of MMC and DEB may have direct implications for the definition of FA phenotype. Another major aspect in ROS formation relies on the extensive evidence pointing to the requirement for oxidative, as well as nitrosative activities in triggering a number of key events in cell division and differentiation, and in early embryogenesis. In turn, antioxidants that may prevent ROS-associated cellular damage in adult cells may prove to exert adverse or fatal outcomes when administered in early life stages. The overall information available on xenobiotic redox biotransformation and on the physiopathological roles of ROS points to the need of addressing ad hoc studies that should take into account the multiplicity of mechanistic events involved.

Thioredoxin suppresses 1-methyl-4-phenylpyridinium-induced neurotoxicity in rat PC12 cells

Thioredoxin (TRX) is a redox-active protein which plays a cytoprotective role against oxidative stress. Geranylgeranylacetone (GGA), used widely as an anti-ulcer drug, has been reported to induce TRX as well as heat shock protein 70 (HSP70) in hepatocytes and other cells. 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), causes dopaminergic denervation and Parkinsonism in humans. The 1-methyl-4-phenylpyridinium ion (MPP +), an active metabolite of MPTP, induces cell death in a rat pheochromocytoma cell line (PC12 cells). We found that MPP + suppresses TRX expression in PC12 cells. Overexpression or administration of TRX attenuates MPP +-induced neurotoxicity on PC12 cells. Moreover, GGA induces expression of TRX and HSP70 and attenuates MPP +-induced toxicity in PC12 cells. These results indicate that TRX and GGA have a possible potential as new therapeutic agents for Parkinson disease.

Thioredoxin overexpression in transgenic mice

Thioredoxin overexpression in transgenic mice

Thioredoxin-mediated redox control of human T cell lymphotropic virus type I (HTLV-I) gene expression

Thioredoxin (TRX) is a small ubiquitous protein with multiple biological functions, including the thiol-mediated redox-regulation of gene expression. We have previously demonstrated that human TRX is overexpressed as a major protein oxidoreductase in human T cell lymphotropic virus type I (HTLV-I)-infected cells. In the present study, we investigated the relationship between TRX and viral gene expression in HTLV-I infection. To study the mechanism that causes overexpression of TRX in HTLV-I-infected cells, we first examined the effect of the HTLV-I transactivator, Tax, on TRX expression. Induction of HTLV-I Tax protein increased the expression of TRX protein in a Tax-transfected Jurkat cell line, JPX-9. Moreover, chloramphenicol acetyltransferase (CAT) analysis with a reporter gene containing the TRX promoter revealed that Tax activates the transcription of TRX gene. To study the role of overexpressed TRX in HTLV-I infection, we next examined the effect of TRX on HTLV-I long terminal repeat (LTR)-mediated transcription using CAT analysis. In an HTLV-I-infected human T cell line MT-2, the HTLV-I LTR transactivation was suppressed by the overexpression of wild-type TRX, but activated by the introduction of inactive mutant TRX. Moreover, in HTLV-I negative Jurkat T cells, the HTLV-I LTR transactivation induced by Tax was also repressed by overexpression of wild-type TRX. Because cellular redox changes were shown to affect the HTLV-I gene expression, it is likely that TRX modulates the HTLV-I gene expression by regulating cellular redox state. Taken together, these findings suggest that overexpressed TRX, which is induced by HTLV-I Tax, may play an important role in HTLV-I infection through the negative regulation of viral gene expression.