Protective roles of redox-active protein thioredoxin-1 for severe acute pancreatitis

Severe acute pancreatitis is a disease with high mortality, and infiltration of inflammatory cells and reactive oxygen species have a crucial role in the pathophysiology of this disease. Thioredoxin-1 (TRX-1) is an endogenous redox-active multifunctional protein with antioxidant and anti-inflammatory effects. TRX-1 is induced in various inflammatory conditions and shows cytoprotective effects. The aim of the present study was to clarify the protective roles of TRX-1 in the host defense mechanism against severe acute pancreatitis. Experimental acute pancreatitis was induced by intraperitoneal administration of cerulein, a CCK analog, and aggravated by lipopolysaccharide injection in transgenic mice overexpressing human TRX-1 (hTRX-1) and control C57BL/6 mice. Transgenic overexpression of hTRX-1 strikingly attenuated the severity of experimental acute pancreatitis. TRX-1 overexpression suppressed neutrophil infiltration as determined by myeloperoxidase activity, oxidative stress as determined by malondialdehyde concentration, and cytoplasmic degradation of inhibitor of kappaB-alpha, thereby suppressing proinflammatory cytokines, tumor necrosis factor-alpha, interleukin-1beta, and interleukin-6; a neutrophil chemoattractant, keratinocyte-derived chemokine; and inducible nitric oxide synthase in the pancreas. Administration of recombinant hTRX-1 also suppressed neutrophil infiltration, reduced the inflammation of the pancreas and the lung, and improved the mortality rate. The present study suggests that TRX-1 has potent antioxidant and anti-inflammatory actions in experimental acute pancreatitis and might be a new therapeutic strategy to improve the prognosis of severe acute pancreatitis.     

Extracellular human thioredoxin-1 inhibits lipopolysaccharide-induced interleukin-1beta expression in human monocyte-derived macrophages

Oxidative stress plays an important role in atherosclerotic vascular disease, and several recent studies were focused on thioredoxin-1 (Trx-1) and its potential protective role against oxidative stress. Since human monocyte-derived macrophages (HMDM) are important cells in several inflammatory diseases including atherosclerosis, we conducted this study to evaluate the impact of extracellular recombinant human Trx-1 (rhTrx-1) on gene expression in lipopolysaccharide-activated HMDM. Our results showed that rhTrx-1 was capable of reducing interleukin (IL)-1beta mRNA and protein synthesis in a dose-dependent manner. This effect was partly mediated through a reduction of NF-kappaB activation as analyzed by transient transfection and gel shift assays. In addition, we showed that the attenuation of NF-kappaB activity was the result of the reduction of both p50 and p65 subunit mRNA and protein synthesis on one hand and of the induction of I-kappaBalpha mRNA and protein expression on the other hand. Moreover, inhibition of endogenous Trx-1 mRNA was also observed, suggesting a contribution to the diminution of NF-kappaB activity since endogenous Trx-1, in contrast to the exogenous Trx-1, activates the NF-kappaB system. Finally, H2O2-oxidized rhTrx-1 reduced IL-1beta mRNA synthesis in lipopolysaccharide-activated HMDM. This result highly suggested that the rhTrx-1 used in this study could be oxidized in the culture medium and, in turn, reduced IL-1beta mRNA and protein synthesis. Taken together, these data indicated a potential new mechanism through which extracellular rhTrx-1 exerts an anti-inflammatory function in HMDM.     

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 （FcεRI） 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 ofcytokines （IL-6 and TNF-α） 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.     

Differential oxidation of thioredoxin-1, thioredoxin-2, and glutathione by metal ions

Metal toxicity often includes the generation of reactive oxygen species (ROS) and subsequent oxidative stress, but whether metals have different effects on the major thiol antioxidant systems is unknown. Here, we examine the effects of arsenic, cadmium, cesium, copper, iron, mercury, nickel, and zinc on glutathione (GSH), cytoplasmic thioredoxin-1 (Trx1), and mitochondrial thioredoxin-2 (Trx2) redox states. GSH/GSSG redox states were determined by HPLC, and Trx1 and Trx2 redox states were determined by Redox Western blot methods. Copper, iron, and nickel showed significant oxidation of GSH but relatively little oxidation of either Trx1 or Trx2. Arsenic, cadmium, and mercury showed little oxidation of GSH but significantly oxidized both Trx1 and Trx2. The magnitude of effects of arsenic, cadmium, and mercury was greater for the mitochondrial Trx2 (>60 mV) compared to the cytoplasmic Trx1 (20 to 40 mV). Apoptosis signal-regulating kinase 1 (ASK1) may be activated by two different pathways, one dependent upon GSH and glutaredoxin and the other independent of GSH and dependent upon thioredoxin. ASK1 activation and cell death were observed with metals that oxidized thioredoxins but not with metals that oxidized GSH. These findings show that metals have differential oxidative effects on the major thiol antioxidant systems and that activation of apoptosis may be associated with metal ions that oxidize thioredoxin and activate ASK1. The differential oxidation of the major thiol antioxidant systems by metal ions suggest that the distinct thiol/disulfide redox couples represented by GSH/GSSG and the thioredoxins may convey different levels of control in apoptotic and toxic signaling pathways.     

Alternative splicing is associated with decreased expression of the redox proto-oncogene thioredoxin-1 in human cancers

Human thioredoxin-1 (Trx-1) is a small redox protein that is overexpressed in a number of human primary tumors, where it is associated with rapid cell proliferation and inhibited apoptosis. Mutation scanning denaturing high-performance liquid chromatography of Trx-1 mRNA in 58 human tumor cell lines found no evidence for changes in the base sequence of human Trx-1 mRNA. An alternatively spliced form of Trx-1 mRNA lacking exons 2 and 3 was found in 7 of the cell lines but it was not translated. The cell lines having the alternatively spliced Trx-1 mRNA had 73% lower total Trx-1 mRNA than the other cell lines, suggesting that alternative splicing may control the level of Trx-1 mRNA in some cancer cells.     

Inhibition of endogenous thioredoxin-1 in the heart of transgenic mice does not confer cardioprotection in ischemic postconditioning

Thioredoxin-1 maintains the cellular redox status and decreases the infarct size in ischemia/reperfusion injury. However, whether the increase of thioredoxin-1 expression or its lack of activity modifies the protection conferred by ischemic postconditioning has not been yet elucidated. The aim was to evaluate if the thioredoxin-1 overexpression enhances the posctconditioning protective effect, and whether the lack of the activity abolishes the reduction of the infarct size. Wild type mice hearts, transgenic mice hearts overexpressing thioredoxin-1, and a dominant negative mutant (C32S/C35S) of thioredoxin-1 were used. The hearts were subjected to 30 min of ischemia and 120 min of reperfusion (Langendorff) (I/R group) or to postconditioning protocol (PostC group). The infarct size in the Wt-PostC group decreased in comparison to the Wt-I/R group (54.6 ± 2.4 vs. 39.2 ± 2.1%, p < 0.05), but this protection was abolished in DN-Trx1-PostC group (49.7 ± 1.1%). The ischemia/reperfusion and postconditioning in mice overexpressing thioredoxin-1 reduced infarct size at the same magnitude (35.9 ± 2.1 and 38.4 ± 1.3%, p < 0.05 vs. Wt-I/R). In Wt-PostC, Trx1-I/R and Trx1- PostC, Akt and GSK3β phosphorylation increased compared to Wt-I/R, without changes in DN-Trx1 groups. In conclusion, given that the cardioprotection conferred by thioredoxin-1 overexpression and postconditioning, is accomplished through the activation of the Akt/GSK3β survival pathway, no synergic effect was evidenced. Thioredoxin-1 plays a key role in the postconditioning, given that when this protein is inactive the cardioprotective mechanism was abolished. Thus, diverse comorbidities or situations modifying the thioredoxin activity, could explain the absence of this strong mechanism of protection in different clinical situations.     

急性低氧对小鼠黏附分子CHL1组织水平表达的影响

目的:观察急性低氧条件下小鼠脑组织及心、肺、肾中黏附分子CHL1的表达变化,为探讨黏附分子CHL1在低氧损伤中的调节作用提供依据.方法:利用本室已建立的小鼠急性低氧模型,将雄性成年C57BL/6小鼠随机分为低氧处理组和常氧对照组(n=10).低氧8h后提取组织蛋白,通过Westem blot检测急性低氧处理后CBL1在...     

Tritrichomonas foetus: pathogenesis of acute infection in normal, estradiol-treated, and stressed mice

Environmental stress and endocrine control can affect pathogenesis of sexually transmitted diseases such as trichomoniasis. Acute Tritrichomonas foetus infection was compared in female BALB/c mice to infections in mice treated with high doses of estradiol or housed in constant bright illumination (stressed). In untreated mice, T. foetus readily colonized the reproductive tract, causing minimal epithelial damage and inflammation. Several fold increases of IFN-gamma, TNF-alpha, MCP-1, and IL-6 cytokines were detected after estradiol-treatment of mice, resulting in greatly enhanced inflammation and tissue damage throughout the reproductive tract. Interestingly, estradiol-treatment of mice resulted in reduced T. foetus colonization compared to untreated mice. Infection in stressed mice resulted in increased tissue damage, inflammation, and inflammatory cytokine expression, although parasite colonization within the reproductive tract was similar to that in untreated mice. These results indicate that either estradiol-treatment or stress result in pathogenesis often observed during severe disease. Alternatively, infection in non-treated mice results in chronic colonization, with little inflammation or pathology.     

The role of thioredoxin-1 in suppression of endoplasmic reticulum stress in Parkinson disease

Endoplasmic reticulum (ER) stress has been implicated in Parkinson disease. We previously reported that thioredoxin 1 (Trx-1) suppressed the ER stress caused by 1-methy-4-phenyl-1,2,3,6-tetrahydropyridine; however, its molecular mechanism remains largely unknown. In the present study, we showed that 1-methyl-4-phenylpyridinium ion (MPP⁺) induced ER stress by activating glucose-regulated protein 78 (GRP78), inositol-requiring enzyme 1α (IRE1α), tumor necrosis factor receptor-associated factor 2 (TRAF2), c-Jun N-terminal kinase (JNK), caspase-12, and C/EBP homologous protein (CHOP) in PC12 cells. The downregulation of Trx-1 aggravated the ER stress and further increased the expression of the above molecules induced by MPP⁺. In contrast, overexpression of Trx-1 attenuated the ER stress and repressed the expression of the above molecules induced by MPP⁺. More importantly, the overexpression of Trx-1 in transgenic mice suppressed ER stress by inhibiting the activation of these molecules. We present, for the first time, the molecular mechanism of Trx-1 suppression of endoplasmic reticulum stress in Parkinson disease in vitro and in vivo. Based on our findings, we conclude that Trx-1 plays a neuroprotective role in Parkinson disease by suppressing ER stress by regulating the activation of GRP78, IRE1α, TRAF2, JNK, caspase-12, and CHOP.     

Low doses of reactive oxygen species protect endothelial cells from apoptosis by increasing thioredoxin-1 expression

The redox regulator thioredoxin-1 (Trx-1) is required for the redox potential of the cell and exerts important functions in cell growth and apoptosis. Severe oxidative stress has been implicated in the oxidation of proteins and cell death. However, the role of low doses of reactive oxygen species (ROS) is poorly understood. Here, we show that 10 and 50 microM H2O2 and short-term exposure to shear stress significantly increased Trx-1 mRNA and protein levels in endothelial cells. Since it is known that Trx-1 exerts anti-apoptotic functions, we next investigated whether low doses of ROS can inhibit basal and serum-depletion induced endothelial cell apoptosis. Indeed, treatment of endothelial cells with 10 and 50 microM H2O2 significantly reduced apoptosis induction. Reduction of Trx-1 expression using an antisense oligonucleotide approach resulted in the induction of apoptosis and abolished the inhibitory effect of low doses of H2O2. Taken together, our results demonstrate that low doses of ROS act as signaling molecules and exert anti-apoptotic functions in endothelial cells via upregulation of the redox-regulator Trx-1.     

Thioredoxin reductase-1 knock down does not result in thioredoxin-1 oxidation

The active site of thioredoxin-1 (Trx1) is oxidized in cells with increased reactive oxygen species (ROS) and is reduced by thioredoxin reductase-1 (TrxR1). The purpose of the present study was to determine the extent to which the redox state of Trx1 is sensitive to changes in these opposing reactions. Trx1 redox state and ROS generation were measured in cells exposed to the TrxR1 inhibitors aurothioglucose (ATG) and monomethylarsonous acid (MMA(III)) and in cells depleted of TrxR1 activity by siRNA knock down. The results showed that all three treatments inhibited TrxR1 activity to similar extents (90% inhibition), but that only MMA(III) exposure resulted in oxidation of Trx1. Similarly, ROS levels were elevated in response to MMA(III), but not in response to ATG or TrxR1 siRNA. Therefore, TrxR1 inhibition alone was not sufficient to oxidize Trx1, suggesting that Trx1-independent pathways should be considered when evaluating pharmacological and toxicological mechanisms involving TrxR1 inhibition.     

Oxidation and nuclear localization of thioredoxin-1 in sparse cell cultures

Reactive oxygen species (ROS) were once viewed only as mediators of toxicity, but it is now recognized that they also contribute to redox signaling through oxidation of specific cysteine thiols on regulatory proteins. Cells in sparse cultures have increased ROS relative to confluent cultures, but it is not known whether protein redox states are affected under these conditions. The purpose of the present study was to determine whether culture conditions affect the redox state of thioredoxin-1 (Trx1), the protein responsible for reducing most oxidized proteins in the cytoplasm and nucleus. The results showed that Trx1 was more oxidized in sparse HeLa cell cultures than in confluent cells. The glutathione pool was also more oxidized, demonstrating that both of the major cellular redox regulating systems were affected by culture density. In addition, the total amount of Trx1 protein was lower and the subcellular distribution of Trx1 was different in sparse cells. Trx1 in sparse cultures was predominantly nuclear whereas it was predominantly cytoplasmic in confluent cultures. This localization pattern was not unique to HeLa cells as it was also observed in A549, Cos-1 and HEK293 cells. These findings demonstrate that Trx1 is subject to changes in expression, redox state and subcellular localization with changing culture density, indicating that the redox environments of the cytoplasm and the nucleus are distinct and have different requirements under different culture conditions.     

Involvement of glutaredoxin-1 and thioredoxin-1 in beta-amyloid toxicity and Alzheimer's disease

Strong evidence indicates oxidative stress in the pathogenesis of Alzheimer's disease (AD). Amyloid beta (Abeta) has been implicated in both oxidative stress mechanisms and in neuronal apoptosis. Glutaredoxin-1 (GRX1) and thioredoxin-1 (TRX1) are antioxidants that can inhibit apoptosis signal-regulating kinase (ASK1). We examined levels of GRX1 and TRX1 in AD brain as well as their effects on Abeta neurotoxicity. We show an increase in GRX1 and a decrease in neuronal TRX1 in AD brains. Using SH-SY5Y cells, we demonstrate that Abeta causes an oxidation of both GRX1 and TRX1, and nuclear export of Daxx, a protein downstream of ASK1. Abeta toxicity was inhibited by insulin-like growth factor-I (IGF-I) and by overexpressing GRX1 or TRX1. Thus, Abeta neurotoxicity might be mediated by oxidation of GRX1 or TRX1 and subsequent activation of the ASK1 cascade. Deregulation of GRX1 and TRX1 antioxidant systems could be important events in AD pathogenesis.     

Suppression of thioredoxin-1 induces premature senescence in normal human fibroblasts

Thioredoxin (TRX) is a ubiquitous multifunctional thiol protein that is critically involved in maintaining cellular redox homeostasis. Levels of thioredoxin-1 (TRX1), the major isoform of TRX, have been shown to correlate with organismal lifespan and age-associated tissue deterioration. Accordingly, we investigated the direct functional effects of suppressing TRX1 levels on cellular senescence, a phenomenon intimately linked with tissue degeneration and aging. Here we find that suppression of TRX1 expression via shRNA rapidly induces premature senescence in young human skin fibroblasts through upregulation of the p53/p21^Cip1/Waf1 and p16^INK4a tumor suppressor pathways. Moreover, inhibition of these pathways by introduction of SV40 Large T Antigen prevents TRX1 suppression-induced premature senescence but not susceptibility to oxidative stressors. Thus our results suggest that TRX1 has a role in suppressing senescence in normal cells in addition to its function as a redox-protective protein.     

Lens-specific regulation of the thioredoxin-1 gene, but not thioredoxin-2, upon in vivo photochemical oxidative stress in the Emory mouse

The angiotensin II (Ang II) type 1 receptor mediates various actions of Ang II, whereas the function of the type 2 (AT2) receptor is not well understood. In the mice lacking the gene encoding the AT2 receptor, the pressor response to Ang II was increased although the underlying mechanism is unknown. We tested the hypothesis that vasoconstrictor response is exaggerated in the AT2 receptor null mice. We measured hemodynamic parameters and evaluated systemic vascular resistance (SVR) in the anesthetized open-chest wild-type and AT2 receptor null mice. Ang II infusion caused dose-dependent increases in SVR in both strains, while the response was significantly higher at 0.5 microgram/kg Ang II in the AT2 receptor null mice (305 +/- 53% of baseline) than in the wild-type mice (179 +/- 27% of baseline). To investigate further the vascular contractility, we examined contraction of aortic rings in vitro. The contraction induced by 1 microM Ang II was increased in the AT2 receptor null mice compared with that in the wild-type mice (0.82 +/- 0.11 vs. 0.54 +/- 0.12 g). Ang II-induced contraction was still greater in the AT2 receptor null mice when calibrated by the maximum tension induced by 90 mM KCl. These data suggest that the AT2 receptor modulates vascular contractility, which may influence blood pressure.     

Ubiquitous expression of mRFP1 in transgenic mice

Fluorescent proteins provide a powerful means to track gene expression and cellular behaviors in the study of model organisms such as mice. Among the new generation of fluorescent protein markers, the monomeric red fluorescent protein mRFP1 is particularly attractive because of its rapid maturation and minimal interference with GFP and GFP-derived markers. Here we evaluate the utility of mRFP1 as a marker in transgenic mice. We show that high level and ubiquitous expression of mRFP1 does not affect mouse development, general physiology, or reproduction. mRFP1 expression can be readily detected with unaided eyes under daylight in transgenic mice on the albino background. The intensity of mRFP1 signals can be used to distinguish homozygous and heterozygous transgenic mice. Together, these features make mRFP1 an attractive marker for broad applications in transgenic research.     

Variable expression of Dkc1 mutations in mice

In humans mutations in DKC1, cause the rare bone marrow failure syndrome dyskeratosis congenita. We have used gene targeting to produce mouse ES cells with Dkc1 mutations that cause DC when in humans. The mutation A353V, the most common human mutation, causes typical DC to very severe DC in humans. Male chimeric mice carrying this mutation do not pass the mutated allele to their offspring. The mutation G402E accounts for a single typical case of DC in a human family. The allele carrying this mutation was transmitted to the offspring with high efficiency. Expression of RNA and protein was reduced compared to wild type animals, but no abnormalities of growth and development or in blood values were found in mutant mice. Thus Dkc1 mutations have variable expression inmice, as in humans. genesis 47:366–373, 2009. © 2009 Wiley‐Liss, Inc.