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Jun-Ichiro Inoue

Publications and source records attributed to Jun-Ichiro Inoue.

At least 19 recordsLinked to original sources
PubMed2006

TNF-receptor associated factor 6-deficient fibroblast is sensitive to the TNF-alpha-induced cell death: involvement of reactive oxygen species.

Tumor necrosis factor receptor (TNFR)-associated factor 6 (TRAF6) has mainly been involved in signaling from CD40 and IL-1 receptor family. While TNF-alpha exerts various biological effects including cell death, the role of TRAF6 in the TNF-alpha signaling remains to be unclear. Here, we demonstrated that murine embryonic fibroblasts (MEFs) derived from TRAF6 knockout (TRAF6KO) mice have increased sensitivity to actinomycin D plus TNF-alpha-induced cell death compared with wild-type MEF. Reactive oxygen species (ROS) were accumulated more in TRAF6KO MEF than in wild-type MEF. An antioxidant, butylated hydroxyanisole (BHA) completely inhibited TNF-alpha-induced cell death and DNA fragmentation. Thus, the TNF-alpha-induced cell death in TRAF6KO MEF was ROS-dependent. Reconstitution of full-length TRAF6 but not N-terminal-deleted TRAF6 constructs in TRAF6KO MEF reversed TNF-alpha-induced cell death, ROS accumulation, and DNA fragmentation completely. Thus, we concluded that resistance against TNF-alpha-induced cell death is rendered by TRAF6, which regulates ROS accumulation.

Animals↗
PubMed2006

Lentivirus vectors expressing short hairpin RNAs against the U3-overlapping region of HIV nef inhibit HIV replication and infectivity in primary macrophages.

Although successful attempts to inhibit HIV-1 replication in T cells using RNAi have been reported, the effect of HIV-specific RNAi on macrophages is not well known. Macrophages are key targets for anti-HIV-1 therapy because they are able to survive long after the initial infection with HIV and can spread the virus to T cells. In this study, we identified a putative RNAi target of HIV, consisting of the portion of the nef gene overlapping the U3 region (Nef366), and generated a lentivirus-based short hairpin RNA (shRNA) expression vector (Lenti shNef366). We show that Lenti shNef366 inhibits (1) HIV-1 replication in a monocytic cell line and in primary monocyte-derived macrophages (MDMs), (2) reactivation of latent HIV-1 infection, and (3) the production of secondary HIV-1 from MDMs harboring a genomic copy of Nef366. Moreover, we found that the up-regulated production of macrophage inflammatory protein 1beta (MIP-1beta), but not MIP-1alpha, in MDMs by Nef expression was considerably suppressed by Lenti shNef366, which suggests that HIV-1 dissemination to T cells through its interaction with HIV-1-infected MDMs can also be controlled by Lenti shNef366. Thus, lentivirus-mediated shRNA expression targeting the U3-overlapping region of HIV nef represents a feasible approach to genetic vaccine therapy for HIV-1.

Cell Line↗
PubMed2006

Inflammatory responses involving tumor necrosis factor receptor-associated factor 6 contribute to in-stent lesion formation in a stent implantation model of rabbit carotid artery.

OBJECTIVE: Inflammatory responses are considered to represent a unique property after stent implantation, and we previously demonstrated that inflammatory signaling involving tumor necrosis factor receptor-associated factor 6 (TRAF6) contributes to neointimal formation in a balloon injury model of rabbit carotid artery. The purpose of this study was to examine the role of TRAF6 in in-stent lesion formation after stent implantation in the rabbit carotid artery. METHODS: Rabbit carotid arteries were injured with a 2F Fogarty catheter, and 28 days later, the same arteries were implanted with a 3-mm-diameter Palmaz-Schatz stent. A dominant negative (DN) form of TRAF6 (pME-FLAG-T6deltaRZ5) was then transferred using a plasmid-based electroporation method. Its effects were evaluated compared with the findings in arteries treated with control plasmid (pME-FLAG). RESULTS: Immunostaining with anti-FLAG tag antibody showed that an expression plasmid vector containing the DN-TRAF6 sequence was successfully transferred to the arterial intima and media. Morphometric analyses revealed that the increase of intimal area in in-stent lesions was significantly inhibited by DN-TRAF6 14 days after stent implantation (DN-TRAF6 group, 3.01 +/- 0.25 x 10(5) microm2 vs control group, 4.25 +/- 0.23 x 10(5) microm2, P < .01), and the cell density was increased compared with that in the control group. In the DN-TRAF6 plasmid-treated vessels, cell replication was prevented in both the intima and media, and fewer leukocytes adhered to the luminal surface. Moreover, DN-TRAF6 suppressed macrophage infiltration, activation of proteases, and proteoglycan accumulation in the in-stent intima. CONCLUSIONS: These findings suggest that TRAF6 plays an important role in cell replication, inflammatory cell infiltration, protease activity, and extracellular matrix accumulation that contributes to in-stent lesion development.

Animals↗
PubMed2005

Identification and characterization of Xenopus laevis homologs of mammalian TRAF6 and its binding protein TIFA.

Tumor necrosis factor receptor (TNFR)-associated factor 6 (TRAF6) transduces signals from members of the TNFR superfamily and the Toll/IL-1R family, leading to activation of transcription factors such as NFkappaB and AP-1. Genetic disruption of the TRAF6 gene in mice results in various developmental abnormalities during embryogenesis, including osteopetrosis, failure of neural tube closure, defective formation of skin appendices, absence of lymph nodes, and absence of mature thymic epithelial cells. To clarify the effect of TRAF6 in development, we previously identified a TRAF-interacting protein with a forkhead-associated domain (TIFA), which binds and activates TRAF6 upon extracellular stimulation. To understand the physiological roles of TRAF6 and TIFA in early development, we studied these genes in Xenopus laevis. Here, we describe identification of X. laevis homologs of mammalian TRAF6 (XTRAF6) and TIFA (XTIFA). As was the case for the mammalian homologs, overexpression of XTRAF6 or XTIFA activated NFkappaB, whereas XTIFA carrying a mutation that abolishes XTRAF6 binding failed to activate NFkappaB, suggesting that XTIFA activates NFkappaB by binding to XTRAF6. XTIFA and XTRAF6 mRNAs were expressed at similar levels in zygotes from the neurula stage and then increased. Whole-mount in situ hybridization revealed that XTRAF6 mRNA was expressed in the head region and neural tube during the neurula stage, and the expression expanded to the pharyngeal apparatus during the tailbud stage. This localization is consistent with the defective neural tube closure and abnormal thymus organogenesis observed in TRAF6-deficient mice. Our results suggest possible cooperation between XTRAF6 and XTIFA during embryogenesis.

Adaptor Proteins, Signal Transducing↗
PubMed2005

Recruitment of tumor necrosis factor receptor-associated factor family proteins to apoptosis signal-regulating kinase 1 signalosome is essential for oxidative stress-induced cell death.

Apoptosis signal-regulating kinase 1 (ASK1) plays a pivotal role in oxidative stress-induced cell death. Reactive oxygen species disrupt the interaction of ASK1 with its cellular inhibitor thioredoxin and thereby activates ASK1. However, the precise mechanism by which ASK1 freed from thioredoxin undergoes oligomerization-dependent activation has not been fully elucidated. Here we show that endogenous ASK1 constitutively forms a high molecular mass complex including Trx ( approximately 1,500-2,000 kDa), which we designate ASK1 signalosome. Upon H(2)O(2) treatment, the ASK1 signalosome forms a higher molecular mass complex at least in part because of the recruitment of tumor necrosis factor receptor-associated factor 2 (TRAF2) and TRAF6. Consistent with our previous findings that TRAF2 and TRAF6 activate ASK1, H(2)O(2)-induced ASK1 activation and cell death were strongly reduced in the cells derived from Traf2-/- and Traf6-/- mice. A novel signaling complex including TRAF2, TRAF6, and ASK1 may thus be the key component in oxidative stress-induced cell death.

Animals↗
PubMed2004

TIFA activates IkappaB kinase (IKK) by promoting oligomerization and ubiquitination of TRAF6.

TRAF6 (tumor necrosis factor receptor-associated factor 6) is a RING (really interesting new gene) domain ubiquitin (Ub) ligase that mediates the activation of protein kinases, such as transforming growth factor beta-activated kinase (TAK1) and IkappaB kinase (IKK), by catalyzing the formation of a unique polyubiquitin chain linked through Lys-63 of Ub. Here, we present evidence that TIFA (TRAF-interacting protein with a forkhead-associated domain, also known as T2BP) activates IKK by promoting the oligomerization and Ub ligase activity of TRAF6. We show that recombinant TIFA protein, but not TRAF6-binding-defective mutant, can activate IKK in crude cytosolic extracts. Furthermore, TIFA activates IKK in an in vitro reconstitution system consisting of purified proteins, including TRAF6, the TAK1 kinase complex, and Ub-conjugating enzyme complex Ubc13-Uev1A. Interestingly, a fraction of recombinant TIFA protein exists as high-molecular-weight oligomers, and only these oligomeric forms of TIFA can activate IKK. Importantly, TIFA induces the oligomerization and polyubiquitination of TRAF6, which leads to the activation of TAK1 and IKK through a proteasome-independent mechanism.

Adaptor Proteins, Signal Transducing↗
PubMed2004

Inflammatory signaling pathway containing TRAF6 contributes to neointimal formation via diverse mechanisms.

OBJECTIVE: The purpose of this study was to investigate the contribution of inflammatory signaling containing tumor necrosis factor receptor-associated factor 6 (TRAF6) to neointimal formation in a balloon injury model of rabbit carotid artery. METHODS: Male Japanese white rabbits fed a normal diet were used. We transferred the dominant negative (DN) form of TRAF6 to a rabbit carotid artery that was subjected to balloon injury by in vivo electroporation method, and then evaluated its effect on intimal lesion formation after balloon injury. RESULTS: An expression plasmid vector containing the TRAF6 DN sequence was successfully transferred to arterial wall cells, and its inhibitory effect on inflammatory signaling was confirmed by the marked suppression of nuclear factor-kappaB (NFkappaB) activity after injury. Morphometric analyses revealed significant inhibition of intimal lesion formation at 7 days after injury. Cell replication and accumulation of macrophages in the media were significantly decreased, and apoptosis was enhanced on day 2. Cell migration to the intima was suppressed on day 4. Extracellular signal-regulated kinase1/2 (ERK1/2) activity at 2 h after injury was also down-regulated. Interestingly, intimal cell replication was significantly blocked when TRAF6 DN was transfected at 7 days after injury. CONCLUSION: TRAF6 plays important roles in cell replication and migration, besides promotion of inflammatory cell infiltration and suppression of apoptosis.

Animals↗
PubMed2004

TGF-beta down-regulates IL-1alpha-induced TLR2 expression in murine hepatocytes.

We have previously reported that the proinflammatory cytokine interleukin (IL)-1alpha can up-regulate functional Toll-like receptor 2 (TLR2) expression in primary-cultured murine hepatocytes, and bacterial lipopeptide (BLP) is capable of signaling through TLR2 to induce serum amyloid A (SAA) expression in hepatocytes. In the present study, we investigated the effect of the anti-inflammatory cytokine transforming growth factor-beta (TGF-beta) on TLR2 expression in primary-cultured murine hepatocytes. At the mRNA and protein levels, TGF-beta up-regulated TLR2 expression but inhibited TLR2 expression induced by IL-1alpha at 24 h. BLP-induced SAA promoter activity could be augmented by pretreatment with IL-1alpha but not TGF-beta or the combination of TGF-beta and IL-1alpha. TLR2 promoter activity and nuclear factor (NF)-kappaB activation by IL-1alpha were inhibited by TGF-beta treatment. Pretreatment with TGF-beta strongly suppressed IL-1alpha-induced TLR2 promoter activity and NF-kappaB activation, which was consistent with the down-regulation of type I IL-1 receptor (IL-1RI) mRNA expression. IL-1alpha up-regulated IL-1RI mRNA, but it was inhibited by the treatment with TGF-beta. These results suggest that TGF-beta suppresses the induction of TLR2 expression by IL-1alpha through down-regulation of IL-1RI expression. These results also demonstrate the disparity between IL-1alpha and TGF-beta in regulating TLR2-mediated SAA production in hepatocytes.

Animals↗
PubMed2004

Traf6 is essential for murine tooth cusp morphogenesis.

Ectodermal appendages such as skin, hair, teeth, and sweat glands are affected in patients with hypohidrotic (anhydrotic) ectodermal dysplasia (HED). It has been established that mutations in the tumor necrosis factor (TNF) superfamily of molecules, i.e., ectodysplasin (EDA), EDA receptor (EDAR), and EDAR-associated death domain (EDARADD; the intracellular adaptor for EDAR), are responsible for several forms of HED in humans and mice. We show here by in situ hybridisation that another TNF family (orphan) receptor, TROY (also known TAJ, TAJ-alpha, TRADE, and TNFRSF19), is strongly coexpressed with Edar in the epithelial enamel knot signalling centres that are believe to regulate cuspal morphogenesis during murine tooth development. Traf6 is known to function as an intracellular adaptor protein for Troy and examination of Traf6 mutant mice revealed abnormalities in molar teeth that are similar but more severe than those produced by mutations in Eda signalling molecules. This finding suggests that, in additional to ectodysplasin, another TNF pathway involving Troy/Traf6 is involved in molar tooth cusp formation and identifies an essential role for a Traf in tooth development. Developmental Dynamics 229:131-135, 2004.

Animals↗
PubMed2003

Epstein-Barr virus latent membrane protein 1 activation of NF-kappaB through IRAK1 and TRAF6.

Epstein-Barr virus latent membrane protein 1 (LMP1) activation of NF-kappaB is critical for Epstein-Barr virus-infected B lymphocyte survival. LMP1 activates the IkappaB kinase complex and NF-kappaB through two cytoplasmic signaling domains that engage tumor necrosis factor receptor-associated factor (TRAF)1/2/3/5 or TRADD and RIP. We now use cells lacking expression of TRAF2, TRAF5, TRAF6, IKKalpha, IKKbeta, IKKgamma, TAB2, IL-1 receptor-associated kinase (IRAK)1, or IRAK4 to assess their roles in LMP1-mediated NF-kappaB activation. LMP1-induced RelA nuclear translocation was similar in IKKalpha knockout (KO) and WT murine embryo fibroblasts (MEFs) but substantially deficient in IKKbeta KO MEFs. NF-kappaB-dependent promoter responses were also substantially deficient in IKKbeta KO MEFs but were hyperactive in IKKalpha KO MEFs. More surprisingly, NF-kappaB responses were near normal in TRAF2 and TRAF5 double-KO MEFs, IKKgamma KO MEFs, TAB2 KO MEFs, and IRAK4 KO MEFs but were highly deficient in TRAF6 KO MEFs and IRAK1 KO HEK293 cells. Consistent with the importance of TRAF6, LMP1-induced NF-kappaB activation in HEK293 cells was inhibited by expression of dominant-negative TAB2 and Ubc13 alleles. These data extend a role for IKKalpha in IKKbeta regulation, identify an unusual IKKbeta-dependent and IKKgamma-independent NF-kappaB activation, and indicate that IRAK1 and TRAF6 are essential for LMP1-induced NF-kappaB activation.

B-Lymphocytes↗
PubMed2003

Transcriptional activity of testis-determining factor SRY is modulated by the Wilms' tumor 1 gene product, WT1.

The Wilms' tumor 1 (WT1) and sex-determining region of the Y chromosome (SRY) genes are essential for development of the mammalian gonads and mutations in these genes are associated with gonadal dysgenesis in humans. The SRY gene encodes a transcription factor with one high-mobility group (HMG) box as a DNA-binding domain. WT1 encodes a transcription factor that contains four contiguous C2H2-type zinc-finger motifs as a DNA/RNA binding or protein-protein interaction domain. Here we report that WT1 binds to and acts synergistically with SRY to activate transcription from a promoter containing SRY-binding sites. This interaction is mediated by the WT1 zinc-finger domain and the SRY HMG box. WT1 mutants associated with Denys-Drash syndrome (DDS), which is characterized by Wilms' tumor, pseudohermaphroditism, and nephropathy, fail to interact with SRY. Wildtype WT1 is recruited to SRY-binding sites in an SRY-dependent manner, whereas DDS mutants are not recruited as efficiently. These results suggest that WT1 forms a complex with SRY to regulate transcription and that this WT1-SRY interaction is important in testis development.

Binding Sites↗
PubMed2003

Cdc2-mediated phosphorylation of Kid controls its distribution to spindle and chromosomes.

The chromokinesin Kid is important in chromosome alignment at the metaphase plate. Here, we report that Kid function is regulated by phosphorylation. We identify Ser427 and Thr463 as M phase-specific phosphorylation sites and Cdc2-cyclin B as a Thr463 kinase. Kid with a Thr463 to alanine mutation fails to be localized on chromosomes and is only detected along spindles, although it retains the ability to bind DNA or chromosomes. Localization of rigor-type mutant Kid, which shows nucleotide-independent microtubule association, is also confined to the spindle, implying that strong association of Kid with the spindle can sequester it from chromosomes. T463A substitution in DNA-binding domain-truncated Kid consistently enhances its spindle localization. At physiological ionic strength, unphosphorylated Kid shows ATP-independent microtubule association, whereas Thr463-phosphorylated Kid shows ATP dependency. Moreover, the stalk region of unphosphorylated Kid interacts with microtubules and the interaction is weakened when Thr463 is phosphorylated. Our data suggest that phosphorylation on Thr463 of Kid downregulates its affinity for microtubules to ensure reversible association with spindles, allowing Kid to bind chromosomes and exhibit its function.

Bacterial Proteins↗
PubMed2003

Identification of TIFA as an adapter protein that links tumor necrosis factor receptor-associated factor 6 (TRAF6) to interleukin-1 (IL-1) receptor-associated kinase-1 (IRAK-1) in IL-1 receptor signaling.

Tumor necrosis factor receptor-associated factor 6 (TRAF6) transduces signals from members of the Toll/interleukin-1 (IL-1) receptor family by interacting with IL-1 receptor-associated kinase-1 (IRAK-1) after IRAK-1 is released from the receptor-MyD88 complex upon IL-1 stimulation. However, the molecular mechanisms underlying regulation of the IRAK-1/TRAF6 interaction are largely unknown. We have identified TIFA, a TRAF-interacting protein with a forkhead-associated (FHA) domain. The FHA domain is a motif known to bind directly to phosphothreonine and phosphoserine. In transient transfection assays, TIFA activates NFkappaBeta and c-Jun amino-terminal kinase. However, TIFA carrying a mutation that abolishes TRAF6 binding or mutations in the FHA domain that are known to abolish FHA domain binding to phosphopeptide fails to activate NFkappaBeta and c-Jun amino-terminal kinase. TIFA, when overexpressed, binds both TRAF6 and IRAK-1 and significantly enhances the IRAK-1/TRAF6 interaction. Furthermore, analysis of endogenous proteins indicates that TIFA associates with TRAF6 constitutively, whereas it associates with IRAK-1 in an IL-1 stimulation-dependent manner in vivo. Thus, TIFA is likely to mediate IRAK-1/TRAF6 interaction upon IL-1 stimulation.

Adaptor Proteins, Signal Transducing↗
PubMed2003

A tetraspanin-family protein, T-cell acute lymphoblastic leukemia-associated antigen 1, is induced by the Ewing's sarcoma-Wilms' tumor 1 fusion protein of desmoplastic small round-cell tumor.

Recurrent chromosomal translocations in neoplasms often generate hybrid genes that play critical roles in tumorigenesis. Desmoplastic small round-cell tumor (DSRCT) is an aggressive malignancy associated with the chromosomal translocation t(11;22)(p13;q12). This translocation generates a chimeric transcription factor, EWS-WT1, which consists of the transcriptional activation domain of the Ewing's sarcoma (EWS) protein and the DNA binding domain of the Wilms' tumor 1 (WT1) protein. One of the splice variants, EWS-WT1(-KTS) lacks three amino acid residues (Lys-Thr-Ser) in the DNA binding domain and transforms NIH3T3 cells. Therefore, it is likely that aberrant gene expression caused by EWS-WT1(-KTS) is involved in the malignant phenotype of DSRCT. Microarray analysis of 9600 human genes revealed that a gene encoding a tetraspanin-family protein, T-cell acute lymphoblastic leukemia-associated antigen 1 (TALLA-1), was induced in EWS-WT1(-KTS)-expressing cell clones. This induction was EWS-WT1(-KTS)-specific, and more importantly, TALLA-1 protein was expressed in the three independent cases of DSRCT. Tetraspanin-family genes encode transmembrane proteins that regulate various cell processes such as cell adhesion, migration and metastasis. Our findings provide a novel insight into the malignant phenotype of DSRCT, suggesting that TALLA-1 is a useful marker for diagnosis and a potential target for the therapy of DSRCT.

Carcinoma, Small Cell↗
PubMed2003

A molecular modeling study of inhibitors of nuclear factor kappa-B (p50)--DNA binding.

Nuclear Factor-kappa B (NF-kappaB) is an inducible transcription factor of the Rel family, and is sequestered in the cytoplasm by the IkappaB family of proteins. NF-kappaB can exist in several dimeric forms, but the p50/p65 heterodimer is the predominant one. Activation of NF-kappaB by a range of stimuli including viral products, and oxidative stress, leads to phosphorylation and proteasome dependent degradation of IkappaB, leading to the release of free NF-kappaB. This free NF-kappaB then binds to its target sites (KB sites in the DNA) to initiate transcription. These kappaB sites are also present in the Long Terminal Repeat (LTR) of HIV-1, and hence NF-kappaB (p50 subunit) binding to LTR-DNA is critical in viral replication. Targeting direct p50-DNA binding, in this regard, is a novel approach to design anti-HIV gene expression inhibitors, which do not have the problem of resistance unlike in other anti-HIV strategies. The present study is a part of our search for leads for the specific inhibition of p50-DNA binding. We have been experimentally studying different types of these inhibitors, and in this work, we attempted to get a common definition of their structural mechanism onto p50-DNA binding. Using three different classes of inhibitors, we modelled their association with the DNA-Binding Region (DBR) of the p50 subunit of NF-kappaB. Docking studies were carried out using a genetic algorithm based program (GOLD). Further, to compare electrostatic complementarity in the association of the inhibitors with the DBR, Molecular Electrostatic Potentials (MEPs) were generated for the DBR and each inhibitor. The results of docking revealed a strong network of hydrogen bonding interactions for every active inhibitor, and the contrary for the less active ones. Further, the MEPs revealed that the DBR of p50 represents a surface of electropositive potential, and the active inhibitors represent a complementary electronegative surface. With the present modelling study we conclude that the principal properties to be possessed by the new leads against p50-DNA binding should be that of having the ability to make a strong network of hydrogen bonds with the DBR of p50, and preferably, having electronegative potentials in their peripheral surface.

Computer Simulation↗
PubMed2003

Down-regulation of TNF-alpha receptors by conophylline in human T-cell leukemia cells.

In the course of our screening for tumor necrosis factor-alpha (TNF-alpha) function inhibitors, conophylline, a vinca alkaloid isolated from the plant Ervatamia microphylla, was found to inhibit TNF-alpha-induced NF-kappaB activation. We studied the effect of conophylline on TNF-alpha-induced NF-kappaB and JNK activations in human T-cell leukemia Jurkat cells. Conophylline inhibited both of these TNF-alpha-induced activations. It also inhibited phosphorylation and degradation of I-kappaB-alpha. Moreover, a receptor binding assay using [125I]-TNF-alpha showed that this inhibitory effect was due to a decrease in the binding of TNF-alpha to the cells. Scatchard analysis of the binding data indicated that conophylline induced only a small change in the affinity of the receptors but a significant change in the receptor number. FACS analysis showed that conophylline reduced the expression of CD120a/TNFR1, the high-affinity receptor for TNF-alpha, on the cell surface. On the other hand, conophylline did not affect the kinetics of internalization and degradation of TNF-alpha/receptor complexes or the half-life of TNF-alpha binding sites. These results indicate that conophylline down-regulates the expression of the TNF-alpha receptors on the cell surface.

Antigens, CD↗