Yasuhiro Gon

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.

p38 Mitogen-Activated Protein Kinase and c-Jun-NH2-Terminal Kinase Regulate RANTES Production by Influenza Virus-Infected Human Bronchial Epithelial Cells

Airway epithelial cells which are the initial site of influenza virus (IV) infection are suggested to participate in airway inflammatory response by expressing various cytokines including RANTES; however, the intracellular signal that regulates RANTES expression has not been determined. In the present study, we examined the role of p38 mitogen-activated protein (MAP) kinase, extracellular signal-regulated kinase (Erk), and c-Jun-NH2-terminal kinase (JNK) in RANTES production by IV-infected human bronchial epithelial cells. The results showed that IV infection induced increases in p38 MAP kinase, and Erk and JNK phosphorylation and activity. SB 203580, PD 98059, and CEP-1347 attenuated IV-infection induced p38 MAP kinase activity, Erk activity, and JNK activity, respectively. SB 203580 and CEP-1347 attenuated RANTES production by 45.3% and 45.2%, respectively, but a combination of these inhibitors additively attenuated by 69.1%. In contrast, PD 98059 did not attenuate. Anti-IL-1α mAb, anti-IL-1β mAb, anti-TNF-α mAb, anti-IL-8 mAb, anti-IFN-β mAb, anti-RANTES mAb, and a combination of these mAbs did not affect IV infection-induced increases in p38 MAP kinase, Erk, and JNK phosphorylation, indicating that each cytokine neutralized by corresponding Ab was not involved in IV infection-induced phosphorylation of MAP kinases. N-acetylcysteine (NAC) did not affect IV infection-induced increases in MAP kinase phosphorylation, whereas NAC attenuated RANTES production by 18.2%, indicating that reactive oxygen species may act as a second messenger leading to RANTES production via p38 MAP kinase- and JNK-independent pathway. These results indicate that p38 MAP kinase and JNK, at least in part, regulate RANTES production by bronchial epithelial cells.

Possible involvement of thioredoxin reductase as well as thioredoxin in cellular sensitivity to cis-diamminedichloroplatinum (II)

The thioredoxin (TRX) system, composed of nicotinamide adenine dinucleotide phosphate (reduced form), TRX, and TRX reductase (TRXR), has multiple biologic functions via thiol-mediated redox control. In this study, we investigated the relationship between intracellular TRXR levels and cellular sensitivity to cis-diamminedichloroplatinum (II) (CDDP). HeLa, a human cervical carcinoma cell line, cultured with CDDP showed a time- and dose-dependent reduction of intracellular TRXR activity, which was well correlated with the decrease in cell viability after exposure to CDDP. In a cell-free system, CDDP was found to directly inactivate the reduced form of purified human TRXR. The CDDP-resistant variants of HeLa cells, established by continuous exposure to CDDP, exhibited an increased expression and activity of TRXR as well as TRX compared with the parental cells. In addition, sodium selenate, an inhibitor of TRXR, was found to increase the susceptibility to CDDP in the CDDP-resistant cells. Moreover, the HeLa cells transfected with an antisense TRXR RNA expression vector to reduce the intracellular enzyme activity displayed an enhanced sensitivity to CDDP. Taken together with previous reports on TRX, these results indicate the possible involvement of TRXR as well as TRX in the cellular sensitivity and resistance to CDDP.

N‐acetylcysteine attenuates TNF‐α‐induced p38 MAP kinase activation and p38 MAP kinase‐mediated IL‐8 production by human pulmonary vascular endothelial cells

We have previously shown that tumour necrosis factor‐α (TNF‐α) activates p38 mitogen‐activated protein (MAP) kinase to produce interleukin‐8 (IL‐8) by human pulmonary vascular endothelial cells. Reactive oxygen species (ROS) including H2O2 generated by TNF‐α can act as signalling intermediates for cytokine induction; therefore, scavenging ROS by anti‐oxidants is important for the regulation of cytokine production. However, the effect of N‐acetylcysteine (NAC), which acts as a precursor of glutathione (GSH) synthesis, on TNF‐α‐induced activation of p38 MAP kinase pathway and p38 MAP kinase‐mediated IL‐8 production by human pulmonary vascular endothelial cells has not been determined. To clarify these issues, we examined the effect of NAC on TNF‐α‐induced activation of p38 MAP kinase, MAP kinase kinase (MKK) 3 and MKK6 which are upstream regulators of p38 MAP kinase, and p38 MAP kinase‐mediated IL‐8 production. Human pulmonary vascular endothelial cells that had been preincubated with NAC were stimulated with TNF‐α and then the activation of p38 MAP kinase and MKK3/MKK6 in the cells and IL‐8 concentrations in the culture supernatants were determined. Intracellular GSH levels increased in NAC‐treated cells. NAC attenuated TNF‐α‐induced activation of p38 MAP kinase and MKK3/MKK6. NAC attenuated p38 MAP kinase‐mediated IL‐8 production by TNF‐α‐stimulated cells. These results indicate that the cellular reduction and oxidation (redox) regulated by intracellular GSH is critical for TNF‐α‐induced activation of p38 MAP kinase pathway and p38 MAP kinase‐mediated IL‐8 production by human pulmonary vascular endothelial cells, and we emphasize that anti‐oxidant therapy is an important strategy for the treatment of acute lung injury.

Proinflammatory cytokine-induced and chemical mediator-induced IL-8 expression in human bronchial epithelial cells through p38 mitogen-activated protein kinase-dependent pathway.

The p38 mitogen-activated protein (MAP) kinase is activated in various cells by proinflammatory cytokines and environmental stresses. However, little is known about the role of p38 MAP kinase in proinflammatory cytokine- and chemical mediator-induced cytokine expression in human bronchial epithelial cells (BECs). In this study we examined the role of p38 MAP kinase in IL-8 expression in BECs to clarify the signal transduction pathway regulating IL-8 expression in BECs stimulated with tumor necrosis factor-alpha (TNF-alpha), IL-1alpha, and platelet-activating factor (PAF). We used TNF-alpha, IL-1alpha, and PAF as inducers for the analysis of the signal transduction pathway and determined IL-8 expression in BECs because TNF-alpha, IL-1alpha, and PAF are known to induce cytokine expression in BECs, and these proinflammatory cytokines and PAF are described to have a role in the production of allergic inflammation. The results showed that TNF-alpha, IL-1alpha, and PAF induced tyrosine phosphorylation of p38 MAP kinase in a dose- and time-dependent manner. The specific p38 MAP kinase inhibitor, SB 203580, completely inhibited TNF-alpha-, IL-1alpha-, or PAF-induced IL-8 protein and mRNA expression in BECs. These results indicated that p38 MAP kinase plays an important role in TNF-alpha-, IL-1alpha-, or PAF-activated signaling pathway, which regulates IL-8 expression in BECs. In addition, these results provide new evidence on a strategy for treatment of airway inflammation with the specific p38 MAP kinase inhibitor.

Tissue remodeling induced by hypersecreted epidermal growth factor and amphiregulin in the airway after an acute asthma attack.

BACKGROUND Epidermal growth factor receptor ligands, such as epidermal growth factor (EGF) and amphiregulin, may play key roles in tissue remodeling in asthma. However, the kinetics of EGF and amphiregulin secretion in the airway after an acute asthma attack and the effect of prolonged airway exposure to these ligands on airway remodeling are unknown. OBJECTIVE To measure the EGF and amphiregulin concentrations in sputa obtained from patients with asthma under various conditions, and to examine the effects of EGF and amphiregulin on the proliferation or differentiation of airway structural cells. METHODS Epidermal growth factor and amphiregulin levels were measured by ELISA in sputum specimens collected from 14 hospitalized children with asthma during an acute asthma attack, 13 stable outpatients with asthma, 8 healthy control children, and 7 children with respiratory tract infections. The effects of EGF and amphiregulin on the proliferation and/or differentiation of normal human bronchial epithelial cells (NHBE), bronchial smooth muscle cells (BSMC), and normal human lung fibroblasts (NHLF) were examined. RESULTS The sputum levels of EGF were significantly higher for about a week after an acute asthma attack compared with the levels in stable subjects with asthma and control subjects. In contrast, upregulation of amphiregulin in the sputa of patients with asthma was observed only during the acute attack. EGF caused proliferation of NHBE, BSMC, and NHLF, whereas amphiregulin induced proliferation of only NHBE. Prolonged exposure of NHBE to EGF and amphiregulin induced mucous cell metaplasia in an IL-13-independent manner. CONCLUSION Acute asthma attacks are associated with hypersecretion of EGF and amphiregulin in the airway. Recurrent acute attacks may aggravate airway remodeling.

Redox Control of Neuronal Damage during Brain Ischemia after Middle Cerebral Artery Occlusion in the Rat: Immunohistochemical and Hybridization Studies of Thioredoxin

Thioredoxin (TRX) is a small, multifunctional protein with a redox-active site and multiple biological functions that include reducing activity for reactive oxygen intermediates. We assayed TRX and TRX mRNA by immunohistochemical methods and hybridization experiments in the rat brain after middle cerebral artery (MCA) occlusion. During ischemia, the immunoreactivity for TRX decreased; it disappeared after MCA occlusion in the ischemic regions. It rapidly decreased and nearly disappeared at 4 and 16 hours after MCA occlusion in the lateral striatum and frontoparietal cortex, respectively. On the other hand, in the perifocal ischemic region, the penumbra, TRX immunoreactivity began to increase 4 hours after MCA occlusion and continued to increase until 24 hours after occlusion. In hybridization experiments, TRX mRNA decreased and nearly disappeared 4 hours after MCA occlusion in the lateral striatum. In the frontoparietal cortex, it decreased until 24 hours after MCA occlusion. In the perifocal ischemic region, TRX mRNA began to increase 4 hours after MCA occlusion and continued to increase until 24 hours. Northern blot analysis showed that total TRX mRNA in the operated hemispheres was induced from 8 hours and increased until 24 hours after the surgical procedures. We previously reported that recombinant TRX promotes the in vitro survival of primary cultured neurons. We now suggest that TRX in the penumbra has neuroprotective functions and that decreased levels of TRX in the ischemic core modify neuronal damage during focal brain ischemia.

ASK1 regulates influenza virus infection-induced apoptotic cell death.

Apoptosis occurs in influenza virus (IV)-infected cells. There are a number of mechanisms for the regulation of apoptosis. However, the molecular mechanism of IV infection-induced apoptosis is still controversial. Apoptosis signal-regulating kinase1 (ASK1) is a ubiquitously expressed mitogen-activated protein kinase kinase kinase (MAPKKK) that activates the SEK1-c-Jun N-terminal kinase (JNK) and MKK3/MKK6-p38 MAPK signaling cascades. ASK1 has been implicated in cytokine- and stress-induced apoptosis. Here, we show the following: (1) IV infection activated ASK1 and concomitantly phosphorylated JNK and p38 MAPK in human bronchial epithelial cells; (2) the activation of JNK and p38 MAPK but not extracellular-regulated kinase (ERK) in embryonic fibroblasts (MEFs) derived from ASK1 knockout mice (ASK1(-/-) MEFs) was depressed compared to MEFs derived from wild type mice (ASK1(+/+) MEFs); and (3) ASK1(-/-) MEFs were defective in IV infection-induced caspase-3 activation and cell death. These results indicate that apoptosis in IV-infected BEC is mediated through ASK1-dependent cascades.

Role of mitogen-activated protein kinases in influenza virus induction of prostaglandin E2 from arachidonic acid in bronchial epithelial cells

Background Influenza virus (IV) infection causes airway inflammation; however, it has not been determined whether IV infection could catabolize arachidonic acid cascade in airway epithelial cells. In addition, the responsible intracellular signalling molecules that catabolize arachidonic acid cascade have not been determined.

Apoptosis signal-regulating kinase 1-mediated signaling pathway regulates hydrogen peroxide-induced apoptosis in human pulmonary vascular endothelial cells

ObjectiveReactive oxygen species initiate pulmonary vascular endothelial cell damage leading to an increase in endothelial permeability resulting in the production of pulmonary edema. Apoptosis signal-regulating kinase (ASK)-1 is a ubiquitously expressed mitogen-activated protein kinase kinase kinase (MAPKKK) that activates the MKK3/MKK6-p38 MAPK and the SEK1-c-Jun N-terminal kinase (JNK) signaling cascade. ASK1 has been implicated in cytokine- and stress-induced apoptosis. However, little is known about the role of ASK1 in apoptosis in hydrogen peroxide (H2O2)-stimulated pulmonary vascular endothelial cells and how ASK1-mediated apoptosis is executed. To clarify this issue, we examined the role of ASK1-p38 MAPK/JNK cascade in apoptosis and caspase-3 activation in H2O2-stimulated pulmonary vascular endothelial cells. DesignExperimental laboratory study. SettingUniversity laboratory. SubjectsNormal human pulmonary artery endothelial cells. InterventionsWestern blot analysis and quantification of apoptosis in cells. Measurements and Main ResultsThe results showed that H2O2 induced ASK1 phosphorylation and concomitantly p38 MAPK and JNK phosphorylation as well as induced caspase-3 activation in pulmonary vascular endothelial cells. To further characterize the role of ASK1 cascade in H2O2-induced apoptosis of pulmonary vascular endothelial cells, the dominant negative form of ASK1-stably transfected porcine artery endothelial cells was used. p38 MAPK and JNK phosphorylation, caspase-3 activation, and apoptosis in the dominant negative form of ASK1-stably transfected porcine artery endothelial cells were depressed compared with those in the parental porcine artery endothelial cells. ConclusionASK1-p38 MAPK/JNK cascade regulates apoptosis of H2O2-stimulated human pulmonary vascular endothelial cells.