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Nonalcoholic steatohepatitis (NASH) is a disorder characterized by simultaneous fat accumulation and chronic inflammation in the liver. In this study, Pin1 expression was revealed to be markedly increased in the livers of mice with methionine choline-deficient (MCD) diet-induced NASH, a rodent model of NASH. In addition, Pin1 KO mice were highly resistant to MCD-induced NASH, based on a series of data showing simultaneous fat accumulation, chronic inflammation, and fibrosis in the liver. In terms of Pin1-induced fat accumulation, it was revealed that the expression levels of peroxisome proliferator-activated receptor α and its target genes were higher in the livers of Pin1 KO mice than in controls. Thus, resistance of Pin1 KO mice to hepatic steatosis is partially attributable to the lack of Pin1-induced down-regulation of peroxisome proliferator-activated receptor α, although multiple other mechanisms are apparently involved. Another mechanism involves the enhancing effect of hematopoietic Pin1 on the expressions of inflammatory cytokines such as tumor necrosis factor and monocyte chemoattractant protein 1 through NF-κB activation, eventually leading to hepatic fibrosis. Finally, to distinguish the roles of hematopoietic or nonhematopoietic Pin1 in NASH development, mice lacking Pin1 in either nonhematopoietic or hematopoietic cells were produced by bone marrow transplantation between wild-type and Pin1 KO mice. The mice having nonhematopoietic Pin1 exhibited fat accumulation without liver fibrosis on the MCD diet. Thus, hepatic Pin1 appears to be directly involved in the fat accumulation in hepatocytes, whereas Pin1 in hematopoietic cells contributes to inflammation and fibrosis. In summary, this is the first study to demonstrate that Pin1 plays critical roles in NASH development. This report also raises the possibility that hepatic Pin1 inhibition to the appropriate level might provide a novel therapeutic strategy for NASH.

DOI： 10.1074/jbc.M112.397133

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The onset and/or growth of breast tumor are controlled, at least in part, by estrogen. Therefore, to prevent the development of breast tumor, estrogen-dependent proliferation of mammary epithelial cells during pregnancy needs to be suppressed once the mammary gland is fully developed to enable lactation. However, the underlying molecular mechanisms remain unknown. Nrk is an X-linked protein serine/threonine kinase in the germinal center kinase family. Herein, we demonstrate a frequent occurrence of breast tumors in homozygous and heterozygous Nrk mutant mice that have experienced pregnancy/parturition. The tumors never developed in the mutant mice without a history of pregnancy/parturition. They exhibited histopathological features of noninvasive tubular adenocarcinoma, and expressed estrogen receptor α. At late gestation when estrogen receptor α expression was significantly reduced in the wild-type mammary gland, grossly normal mammary glands in the pregnant Nrk mutant mice occasionally contained hyperplastic foci continuously expressing the receptor. Consistently, Nrk expression was induced in the wild-type mammary gland at this period of pregnancy. On the other hand, the pregnant Nrk mutant mice also showed elevated blood estrogen levels at late gestation. We suggest that Nrk suppresses the excessive proliferation of mammary epithelial cells during pregnancy, and the impairment of this regulatory system leads to frequent occurrence of breast tumor in Nrk mutant mice.

DOI： 10.1016/j.ajpath.2016.06.005

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AMP-activated protein kinase (AMPK) plays a critical role in metabolic regulation. In this study, first, it was revealed that Pin1 associates with any isoform of γ, but not with either the α or the β subunit, of AMPK. The association between Pin1 and the AMPK γ1 subunit is mediated by the WW domain of Pin1 and the Thr(211)-Pro-containing motif located in the CBS domain of the γ1 subunit. Importantly, overexpression of Pin1 suppressed AMPK phosphorylation in response to either 2-deoxyglucose or biguanide stimulation, whereas Pin1 knockdown by siRNAs or treatment with Pin1 inhibitors enhanced it. The experiments using recombinant Pin1, AMPK, LKB1, and PP2C proteins revealed that the protective effect of AMP against PP2C-induced AMPKα subunit dephosphorylation was markedly suppressed by the addition of Pin1. In good agreement with the in vitro data, the level of AMPK phosphorylation as well as the expressions of mitochondria-related genes, such as PGC-1α, which are known to be positively regulated by AMPK, were markedly higher with reduced triglyceride accumulation in the muscles of Pin1 KO mice as compared with controls. These findings suggest that Pin1 plays an important role in the pathogenic mechanisms underlying impaired glucose and lipid metabolism, functioning as a negative regulator of AMPK.

DOI： 10.1074/jbc.M115.658559

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Pin1 is a unique regulator, which catalyzes the conversion of a specific phospho-Ser/Thr-Pro-containing motif in target proteins. Herein, we identified CRTC2 as a Pin1-binding protein by overexpressing Pin1 with Myc and FLAG tags in mouse livers and subsequent purification of the complex containing Pin1. The association between Pin1 and CRTC2 was observed not only in overexpression experiments but also endogenously in the mouse liver. Interestingly, Ser(136) in the nuclear localization signal of CRTC2 was shown to be involved in the association with Pin1. Pin1 overexpression in HepG2 cells attenuated forskolin-induced nuclear localization of CRTC2 and cAMP-responsive element (CRE) transcriptional activity, whereas gene knockdown of Pin1 by siRNA enhanced both. Pin1 also associated with CRTC1, leading to their cytosol localization, essentially similar to the action of CRTC2. Furthermore, it was shown that CRTC2 associated with Pin1 did not bind to CREB. Taken together, these observations indicate the association of Pin1 with CRTC2 to decrease the nuclear CBP·CRTC·CREB complex. Indeed, adenoviral gene transfer of Pin1 into diabetic mice improved hyperglycemia in conjunction with normalizing phosphoenolpyruvate carboxykinase mRNA expression levels, which is regulated by CRE transcriptional activity. In conclusion, Pin1 regulates CRE transcriptional activity, by associating with CRTC1 or CRTC2.

DOI： 10.1074/jbc.M110.137836

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Prolonged stimulation of FRTL-5 thyroid cells with cAMP-generating agents including thyroid-stimulating hormone (TSH) or cAMP analogues potentiates tyrosine phosphorylation of insulin receptor substrate (IRS)-2 triggered by insulin-like growth factor (IGF)-I, leading to enhancement of IGF-I-dependent proliferation. Because we identified HSP90 as an IRS-2-interacting protein, the roles of HSP90 in potentiation of IGF signals through IRS-2 were investigated. We found that prolonged dibutyryl cAMP treatment induced serine/threonine phosphorylation of IRS-2. Using a specific inhibitor of HSP90 chaperone activity, geldanamycin, or small interfering RNA against HSP90, we showed that HSP90 mediates cAMP-induced serine/threonine phosphorylation of IRS-2. Furthermore, inhibition of HSP90 by geldanamycin during dibutyryl cAMP pretreatment of cells for 24h suppressed cAMP-dependent potentiation of tyrosine phosphorylation of IRS-2 induced by IGF-I. Taking together, we conclude that HSP90 interacting with IRS-2 mediates cAMP-dependent serine/threonine phosphorylation of IRS-2 via its chaperone activity, leading to potentiation of tyrosine phosphorylation of IRS-2 induced by IGF-I.

DOI： 10.1016/j.mce.2011.06.029

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DOI： 10.1111/cen.12317

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Insulin receptor substrates (IRSs) are known to play important roles in mediating intracellular insulin-like growth factors (IGFs)/insulin signaling. In this study, we identified components of messenger ribonucleoprotein (mRNP) as IRS-1-associated proteins. IRS-1 complex formation analysis revealed that IRS-1 is incorporated into the complexes of molecular mass more than 1000 kDa, which were disrupted by treatment with RNase. Furthermore, oligo(dT) beads precipitated IRS-1 from cell lysates, showing that the IRS-1 complexes contained messenger RNA. Taken together with the data that IRS-1 was fractionated into the polysome-containing high-density fractions, we concluded that IRS-1 forms the novel complexes with mRNPs.

DOI： 10.1016/j.febslet.2013.05.066

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OBJECTIVE: Resistin-like molecule (RELM) β is a secretory protein homologous to resistin and reportedly contributes to local immune response regulation in gut and bronchial epithelial cells. However, we found that activated macrophages also express RELMβ and thus investigated the role of RELMβ in the development of atherosclerosis. APPROACH AND RESULTS: It was demonstrated that foam cells in atherosclerotic lesions of the human coronary artery abundantly express RELMβ. RELMβ knockout ((-/-)) and wild-type mice were mated with apolipoprotein E-deficient background mice. RELMβ(-/-) apolipoprotein E-deficient mice exhibited less lipid accumulation in the aortic root and wall than RELMβ(+/+) apolipoprotein E-deficient mice, without significant changes in serum lipid parameters. In vitro, RELMβ(-/-) primary cultured peritoneal macrophages (PCPMs) exhibited weaker lipopolysaccharide-induced nuclear factor-κB classical pathway activation and inflammatory cytokine secretion than RELMβ(+/+), whereas stimulation with RELMβ upregulated inflammatory cytokine expressions and increased expressions of many lipid transporters and scavenger receptors in PCPMs. Flow cytometric analysis revealed inflammatory stimulation-induced RELMβ in F4/80(+) CD11c(+) PCPMs. In contrast, the expressions of CD11c and tumor necrosis factor were lower in RELMβ(-/-) PCPMs, but both were restored by stimulation with recombinant RELMβ. CONCLUSIONS: RELMβ is abundantly expressed in foam cells within plaques and contributes to atherosclerosis development via lipid accumulation and inflammatory facilitation.

DOI： 10.1161/ATVBAHA.113.301546

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Continuous stimulation of cells with insulin-like growth factors (IGFs) in G(1) phase is a well established requirement for IGF-induced cell proliferation; however, the molecular components of this prolonged signaling pathway that is essential for cell cycle progression from G(1) to S phase are unclear. IGF-I activates IGF-I receptor (IGF-IR) tyrosine kinase, followed by phosphorylation of substrates such as insulin receptor substrates (IRS) leading to binding of signaling molecules containing SH2 domains, including phosphatidylinositol 3-kinase (PI3K) to IRS and activation of the downstream signaling pathways. In this study, we found prolonged (>9 h) association of PI3K with IGF-IR induced by IGF-I stimulation. PI3K activity was present in this complex in thyrocytes and fibroblasts, although tyrosine phosphorylation of IRS was not yet evident after 9 h of IGF-I stimulation. IGF-I withdrawal in mid-G(1) phase impaired the association of PI3K with IGF-IR and suppressed DNA synthesis the same as when PI3K inhibitor was added. Furthermore, we demonstrated that Tyr(1316)-X-X-Met of IGF-IR functioned as a PI3K binding sequence when this tyrosine is phosphorylated. We then analyzed IGF signaling and proliferation of IGF-IR(-/-) fibroblasts expressing exogenous mutant IGF-IR in which Tyr(1316) was substituted with Phe (Y1316F). In these cells, IGF-I stimulation induced tyrosine phosphorylation of IGF-IR and IRS-1/2, but mutated IGF-IR failed to bind PI3K and to induce maximal phosphorylation of GSK3β and cell proliferation in response to IGF-I. Based on these results, we concluded that PI3K activity bound to IGF-IR, which is continuously sustained by IGF-I stimulation, is required for IGF-I-induced cell proliferation.

DOI： 10.1074/jbc.M112.393074

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BACKGROUND: IGFs play key roles in intrauterine and postnatal growth through the IGF-I receptor (IGF-IR). We identified a family bearing a new heterozygous missense mutation at the L2 domain of IGF-IR (R431L). METHOD: We analysed the nucleotide sequences of the IGF1R gene of the family. We prepared R(-) cells (fibroblasts with targeted disruption of the IGF-IR gene) expressing wild-type or R431L IGF-IR and performed functional analyses by evaluating IGF-I binding, IGF-I-stimulated DNA synthesis, tyrosine phosphorylation of IGF-IR and its substrates, and internalization by measuring [(125) I]IGF-I internalization. We also performed confocal microscopy analysis. RESULTS: We identified a family bearing a new heterozygous missense mutation at the L2 domain of IGF-IR (R431L) through an 8-year-old girl and her mother, both born with intrauterine growth retardation. In experiments conducted using cells homozygously transfected with the IGF-IR R431L mutation; (i) IGF-I binding was not affected; (ii) DNA synthesis induced by IGF-I was decreased; (iii) IGF-IR internalization stimulated by IGF-I was decreased and (iv) IGF-I-stimulated tyrosine phosphorylation was reduced IGF-IR by low concentrations of IGF-I and on insulin receptor substrate (IRS)-1 and IRS-2. CONCLUSION: A missense mutation (R431L) leads to the inhibition of cell proliferation, attenuation of IGF signalling and decrease in internalization of IGF-IR. The results of this study suggest a novel link between a mutation at the IGF-IR L2 domain and intrauterine and postnatal growth retardation.

DOI： 10.1111/j.1365-2265.2012.04357.x

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Pin1 and Par14 are parvulin-type peptidyl-prolyl cis/trans isomerases. Although numerous proteins have been identified as Pin1 substrates, the target proteins of Par14 remain largely unknown. Par14 expression levels are increased in the livers and embryonic fibroblasts of Pin1 KO mice, suggesting a compensatory relationship between the functions of Pin1 and Par14. In this study, the association of Par14 with insulin receptor substrate 1 (IRS-1) was demonstrated in HepG2 cells overexpressing both as well as endogenously in the mouse liver. The analysis using deletion-mutated Par14 and IRS-1 constructs revealed the N-terminal portion containing the basic domain of Par14 and the two relatively C-terminal portions of IRS-1 to be involved in these associations, in contrast to the WW domain of Pin1 and the SAIN domain of IRS-1. Par14 overexpression in HepG2 markedly enhanced insulin-induced IRS-1 phosphorylation and its downstream events, PI3K binding with IRS-1 and Akt phosphorylation. In contrast, treating HepG2 cells with Par14 siRNA suppressed these events. In addition, overexpression of Par14 in the insulin-resistant ob/ob mouse liver by adenoviral transfer significantly improved hyperglycemia with normalization of hepatic PEPCK and G6Pase mRNA levels, and gene suppression of Par14 using shRNA adenovirus significantly exacerbated the glucose intolerance in Pin1 KO mice. Therefore, although Pin1 and Par14 associate with different portions of IRS-1, the prolyl cis/trans isomerization in multiple sites of IRS-1 by these isomerases appears to be critical for efficient insulin receptor-induced IRS-1 phosphorylation. This process is likely to be one of the major mechanisms regulating insulin sensitivity and also constitutes a potential therapeutic target for novel insulin-sensitizing agents.

DOI： 10.1074/jbc.M113.485730

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Accumulated evidence indicates that oxidative stress causes and/or promotes insulin resistance; however, the mechanism by which this occurs is not fully understood. This study was undertaken to elucidate the molecular mechanism by which oxidative stress induced by paraquat impairs insulin-dependent glucose uptake in 3T3-L1 adipocytes. We confirmed that paraquat-induced oxidative stress decreased glucose transporter 4 (GLUT4) translocation to the cell surface, resulting in repression of insulin-dependent 2-deoxyglucose uptake. Under these conditions, oxidative stress did not affect insulin-dependent tyrosine phosphorylation of insulin receptor, insulin receptor substrate (IRS)-1 and -2, or binding of the phosphatidylinositol 3'-OH kinase (PI 3-kinase) p85 regulatory subunit or p110alpha catalytic subunit to each IRS. In contrast, we found that oxidative stress induced by paraquat inhibited activities of PI 3-kinase bound to IRSs and also inhibited phosphorylation of Akt, the downstream serine/threonine kinase that has been shown to play an essential role in insulin-dependent translocation of GLUT4 to the plasma membrane. Overexpression of active form Akt (myr-Akt) restored inhibition of insulin-dependent glucose uptake by paraquat, indicating that paraquat-induced oxidative stress inhibits insulin signals upstream of Akt. Paraquat treatment with and without insulin treatment decreased the activity of class Ia PI 3-kinases p110alpha and p110beta that are mainly expressed in 3T3-L1 adipocytes. However, paraquat treatment did not repress the activity of the PI 3-kinase p110alpha mutated at Cys(90) in the p85 binding region. These results indicate that the PI 3-kinase p110 is a possible primary target of paraquat-induced oxidative stress to reduce the PI 3-kinase activity and impaired glucose uptake in 3T3-L1 adipocytes.

DOI： 10.1074/jbc.M110.126482

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Bioactivities of IGFs in various cells are often potentiated in the presence of other hormones. In previous studies we showed that pretreatment of rat FRTL-5 thyroid cells with TSH or other cAMP-generating agents markedly potentiated DNA synthesis induced by IGF-I. Under these conditions we found that phosphatidylinositol (PI) 3-kinase was activated in response to either cAMP or IGF stimulus, and both activation modes were indispensable for the potentiation of DNA synthesis. The present studies were undertaken to elucidate how cAMP and/or IGF-I stimulus regulated the G1 cyclin-cyclin dependent kinase (CDK)-inhibitor system, and to determine the roles of PI 3-kinase activation by cAMP or IGF-I stimulus in this system. We found that cAMP pretreatment enhanced IGF-I-dependent increases in cyclin D1, due to synergistic increases in mRNA and elevation of translation rates. Furthermore, cAMP pretreatment enhanced IGF-I-induced protein degradation of the CDK inhibitor, p27(Kip1). These changes well explained an increase in cyclin E, leading to marked activation of G1 CDKs, followed by retinoblastoma protein phosphorylation. Our results using a PI 3-kinase inhibitor showed that cAMP-dependent PI 3-kinase activation plays an important role in the increase in cyclin D1 translation. In contrast, IGF-I-dependent PI 3-kinase activation was required for the increase in cyclin D1 mRNA levels and degradation of p27(Kip1). Together, the present study elucidates the role of cAMP and IGF-I in differentially activating PI 3-kinase as a mediator of multiple molecular events. These events converge in the regulation of cyclin D1 and p27(Kip1), leading to cAMP-dependent potentiation of IGF-I-dependent CDK activation and DNA synthesis.

DOI： 10.1210/en.2007-1443

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DOI： 10.1016/j.placenta.2024.03.013

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Insulin receptor substrates (IRSs) play central roles in insulin/insulin-like growth factor (IGF) signaling and mediate a variety of their bioactivities. IRSs are tyrosine-phosphorylated by activated insulin receptor/IGF-I receptor tyrosine kinase in response to insulin/IGF, and are recognized by signaling molecules possessing the SH2 domain such as phosphatidylinositol 3-kinase (PI3K), leading to the activation of downstream pathways. Recent studies have suggested that degradation of IRSs by the ubiquitin-proteasome pathway leads to impaired insulin/IGF signaling, but the precise mechanism underlying the process is still unclear. In this study, we identified deubiquitinating enzyme ubiquitin specific protease 7 (USP7) as an IRS-2-interacting protein and demonstrated that deubiquitinase activity of USP7 plays important roles in IRS-2 stabilization through the ubiquitin-proteasome pathway. In addition, insulin treatment dissociated USP7 from IRS-2, leading to degradation of IRS-2. This dissociation was prevented by treatment with LY294002, a PI3K inhibitor, indicating that insulin activation of the PI3K pathway leads to dissociation of IRS-2 from USP7 and IRS-2 degradation. We obtained similar results for IRS-1 in cells treated with insulin and for IRS-2 in cells treated with IGF-I. Taken together, this is the first report demonstrating that USP7 is an IRS-1/2 deubiquitinating enzyme forming a negative feedback loop in insulin/IGF signaling.

DOI： 10.1016/j.bbrc.2012.05.093

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We previously demonstrated that long-term pretreatment of rat FRTL-5 thyroid cells with TSH or cAMP-generating reagents potentiated IGF-I-dependent DNA synthesis. Under these conditions, cAMP treatment increased tyrosine phosphorylation of a 125-kDa protein (p125) and its association with a p85 regulatory subunit of phosphatidylinositol 3-kinase (p85 PI3K), which were suggested to mediate potentiation of DNA synthesis. This study was undertaken to identify p125 and to elucidate its roles in potentiation of DNA synthesis induced by IGF-I. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis revealed p125 to be a rat ortholog of human XB130, which we named PI3K-associated protein (PI3KAP). cAMP treatment elevated PI3KAP/XB130 mRNA and protein levels as well as tyrosine phosphorylation and interaction with p85 PI3K leading to increased PI3K activities associated with PI3KAP/XB130, supporting the role of PI3KAP/XB130 in DNA synthesis potentiation. Importantly, PI3KAP/XB130 knockdown attenuated cAMP-dependent potentiation of IGF-I-induced DNA synthesis. Furthermore, c-Src was associated with PI3KAP/XB130 and was activated in response to cAMP. Addition of Src family kinase inhibitors, PP1 or PP2, during cAMP treatment abolished tyrosine phosphorylation of PI3KAP/XB130 and its interaction with p85 PI3K. Finally, introduction of PI3KAP/XB130 into NIH3T3 fibroblasts lacking endogenous PI3KAP/XB130 enhanced IGF-I-induced DNA synthesis; however, a mutant Y72F incapable of binding to p85 PI3K did not show this response. Together, these data indicate that cAMP-dependent induction of PI3KAP/XB130, which is associated with PI3K, is required for enhancement of IGF mitogenic activities.

DOI： 10.1210/me.2011-1349

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Peptidyl-prolyl cis/trans isomerase NIMA-interacting 1 (Pin1) is a unique enzyme that associates with the pSer/Thr-Pro motif and catalyzes cis-trans isomerization. We identified Pin1 in the immunoprecipitates of overexpressed IRS-1 with myc and FLAG tags in mouse livers and confirmed the association between IRS-1 and Pin1 by not only overexpression experiments but also endogenously in the mouse liver. The analysis using deletion- and point-mutated Pin1 and IRS-1 constructs revealed the WW domain located in the N terminus of Pin1 and Ser-434 in the SAIN (Shc and IRS-1 NPXY binding) domain of IRS-1 to be involved in their association. Subsequently, we investigated the role of Pin1 in IRS-1 mediation of insulin signaling. The overexpression of Pin1 in HepG2 cells markedly enhanced insulin-induced IRS-1 phosphorylation and its downstream events: phosphatidylinositol 3-kinase binding with IRS-1 and Akt phosphorylation. In contrast, the treatment of HepG2 cells with Pin1 siRNA or the Pin1 inhibitor Juglone suppressed these events. In good agreement with these in vitro data, Pin1 knock-out mice exhibited impaired insulin signaling with glucose intolerance, whereas adenoviral gene transfer of Pin1 into the ob/ob mouse liver mostly normalized insulin signaling and restored glucose tolerance. In addition, it was also demonstrated that Pin1 plays a critical role in adipose differentiation, making Pin1 knock-out mice resistant to diet-induced obesity. Importantly, Pin1 expression was shown to be up-regulated in accordance with nutrient conditions such as food intake or a high-fat diet. Taken together, these observations indicate that Pin1 binds to IRS-1 and thereby markedly enhances insulin action, essential for adipogenesis.

DOI： 10.1074/jbc.M110.206904

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DOI： 10.1101/2025.05.27.656518

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Nascent cargo proteins in the endoplasmic reticulum are transported to the Golgi by COPII carriers. Typical COPII vesicles are 60-70 nm in diameter, and much larger macromolecules, such as procollagen, are transported by atypical large COPII carriers in mammalian cells. The formation of large COPII carriers is enhanced by Cul3 ubiquitin ligase, which mono-ubiquitinates Sec31A, a COPII coat protein. However, the deubiquitinating enzyme for Sec31A was unclear. Here, we show that the deubiquitinating enzyme USP8 interacts with and deubiquitinates Sec31A. The interaction was mediated by the adaptor protein STAM1. USP8 overexpression inhibited the formation of large COPII carriers. By contrast, USP8 knockdown caused the accumulation of COPII coat proteins around the cis-Golgi, promoted the intracellular trafficking of procollagen IV from the endoplasmic reticulum to the Golgi, and increased collagen IV secretion. We concluded that USP8 deubiquitinates Sec31A and inhibits the formation of large COPII carriers, thereby suppressing collagen IV secretion.

DOI： 10.1016/j.bbrc.2018.03.202

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The dynamic process of adipose differentiation involves stepwise expressions of transcription factors and proteins specific to the mature fat cell phenotype. In this study, it was revealed that expression levels of IntS6 and IntS11, subunits of the Integrator complex, were increased in 3T3-L1 cells in the period when the cells reached confluence and differentiated into adipocytes, while being reduced to basal levels after the completion of differentiation. Suppression of IntS6 or IntS11 expression using siRNAs in 3T3-L1 preadipocytes markedly inhibited differentiation into mature adipocytes, based on morphological findings as well as mRNA analysis of adipocyte-specific genes such as Glut4, perilipin and Fabp4. Although Pparγ2 protein expression was suppressed in IntS6 or IntS11-siRNA treated cells, adenoviral forced expression of Pparγ2 failed to restore the capacity for differentiation into mature adipocytes. Taken together, these findings demonstrate that increased expression of Integrator complex subunits is an indispensable event in adipose differentiation. Although further study is necessary to elucidate the underlying mechanism, the processing of U1, U2 small nuclear RNAs may be involved in cell differentiation steps.

DOI： 10.1016/j.bbrc.2013.03.029

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Glucose transporter 1 (GLUT1) is widely distributed throughout various tissues and contributes to insulin-independent basal glucose uptake. Using a split-ubiquitin membrane yeast two-hybrid system, we newly identified 4F2 heavy chain (4F2hc) as a membrane protein interacting with GLUT1. Though 4F2hc reportedly forms heterodimeric complexes between amino acid transporters, such as LAT1 and LAT2, and regulates amino acid uptake, we investigated the effects of 4F2hc on GLUT1 expression and the associated glucose uptake. First, FLAG-tagged 4F2hc and hemagglutinin-tagged GLUT1 were overexpressed in human embryonic kidney 293 cells and their association was confirmed by coimmunoprecipitation. The green fluorescent protein-tagged 4F2hc and DsRed-tagged GLUT1 showed significant, but incomplete, colocalization at the plasma membrane. In addition, an endogenous association between GLUT1 and 4F2hc was demonstrated using mouse brain tissue and HeLa cells. Interestingly, overexpression of 4F2hc increased the amount of GLUT1 protein in HeLa and HepG2 cells with increased glucose uptake. In contrast, small interfering RNA (siRNA)-mediated 4F2hc gene suppression markedly reduced GLUT1 protein in both cell types, with reduced glucose uptake. While GLUT1 mRNA levels were not affected by overexpression or gene silencing of 4F2hc, GLUT1 degradation after the addition of cycloheximide was significantly suppressed by 4F2hc overexpression and increased by 4F2hc siRNA treatment. Taken together, these observations indicate that 4F2hc is likely to be involved in GLUT1 stabilization and to contribute to the regulation of not only amino acid but also glucose metabolism.

DOI： 10.1152/ajpcell.00416.2010

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Insulin-like growth factors (IGFs) induce proliferation of various cell types and play important roles in somatic growth and cancer development. Phosphorylation of insulin receptor substrate (IRS)-1/2 by IGF-I receptor tyrosine kinase is essential for IGF action. Here we identify Nedd4 as an IRS-2 ubiquitin ligase. Nedd4 monoubiquitinates IRS-2, which promotes its association with Epsin1, a ubiquitin-binding protein. Nedd4 recruits IRS-2 to the membrane, probably through promoting Epsin1 binding, and enhances IGF-I receptor-induced IRS-2 tyrosine phosphorylation. In thyroid FRTL-5 cells, activation of the cyclic AMP pathway increases the association of Nedd4 with IRS-2, thereby enhancing IRS-2-mediated signalling and cell proliferation induced by IGF-I. The Nedd4 and IRS-2 association is also required for maximal activation of IGF-I signalling and cell proliferation in prostate cancer PC-3 cells. Nedd4 overexpression accelerates zebrafish embryonic growth through IRS-2 in vivo. We conclude that Nedd4-induced monoubiquitination of IRS-2 enhances IGF signalling and mitogenic activity.

DOI： 10.1038/ncomms7780

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Language：Japanese   Publishing type：Research paper (scientific journal)   Publisher：Rockefeller University Press  

K63-linked ubiquitin chains attached to plasma membrane proteins serve as tags for endocytosis and endosome-to-lysosome sorting. USP8 is an essential deubiquitinase for the maintenance of endosomal functions. Prolonged depletion of USP8 leads to cell death, but the major effects on cellular signaling pathways are poorly understood. Here, we show that USP8 depletion causes aberrant accumulation of K63-linked ubiquitin chains on endosomes and induces immune and stress responses. Upon USP8 depletion, two different decoders for K63-linked ubiquitin chains, TAB2/3 and p62, were recruited to endosomes and activated the TAK1–NF-κB and Keap1–Nrf2 pathways, respectively. Oxidative stress, an environmental stimulus that potentially suppresses USP8 activity, induced accumulation of K63-linked ubiquitin chains on endosomes, recruitment of TAB2, and expression of the inflammatory cytokine. The results demonstrate that USP8 is a gatekeeper of misdirected ubiquitin signals and inhibits immune and stress response pathways by removing K63-linked ubiquitin chains from endosomes.

DOI： 10.1083/jcb.202306013

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Nik-related kinase (Nrk) is a member of the germinal center kinase IV family and suppresses Akt signaling. In vivo, Nrk prevents placental hyperplasia and breast cancer formation. Here, we show that Nrk is regulated by the chaperone-dependent ubiquitin ligase carboxyl terminus of heat-shock protein (Hsp)70-interacting protein (CHIP). Immunoprecipitation and liquid chromatography-tandem mass spectrometry analysis reveal that Nrk preferentially interacts with CHIP and Hsp70/90 family proteins. Nrk protein levels are decreased by CHIP overexpression and increased by siRNA-mediated CHIP knockdown. Our results indicate that Nrk is ubiquitinated by CHIP in a chaperone-dependent manner, resulting in its proteasomal degradation. CHIP targets a fraction of Nrk molecules that have lost the ability to regulate Akt signaling. We conclude that CHIP plays an important role in regulating Nrk protein levels.

DOI： 10.1002/1873-3468.13769

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In skeletal muscle, sortilin plays a predominant role in the sorting of glucose transporter 4 (Glut4), thereby controlling glucose uptake. Moreover, our previous study suggested that the sortilin expression levels are also implicated in myogenesis. Despite the importance of sortilin in skeletal muscle, however, the regulation of sortilin expression has not been completely understood. In the present study, we analyzed if the sortilin expression is regulated by glucose in C2C12 myocytes and rat skeletal muscles in vivo. Sortilin protein expression was elevated upon C2C12 cell differentiation and was further enhanced in the presence of a high concentration of glucose. The gene expression and protein degradation of sortilin were not affected by glucose. On the other hand, rapamycin partially reduced sortilin induction by a high concentration of glucose, which suggested that sortilin translation could be regulated by glucose, at least in part. We also examined if the sortilin regulation by glucose was also observed in skeletal muscles that were obtained from fed or fasted rats. Sortilin expression in both gastrocnemius and extensor digitorum longus (EDL) muscle was significantly decreased by 17-18h of starvation. On the other hand, pathological levels of high blood glucose did not alter the sortilin expression in rat skeletal muscle. Overall, the present study suggests that sortilin protein levels are reduced under hypoglycemic conditions by post-transcriptional control in skeletal muscles.

DOI： 10.1507/endocrj.EJ16-0319

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Insulin receptor substrates (IRSs) are phosphorylated by IGF-I receptor tyrosine kinase in a ligand-dependent manner. In turn, they bind to and activate effector proteins such as PI3K, leading to various cell responses including cell proliferation. We had reported that ubiquitin ligase Nedd4 induces mono-ubiquitination of IRS-2, thereby enhancing IRS-2 tyrosine phosphorylation, leading to increased IGF signaling and mitogenic activity. Here we show that ubiquitin-specific protease 15 (USP15) antagonizes the effect of Nedd4 on IRS-2. We identified USP15 as a protein that preferentially bound to IRS-2 when IRS-2 was conjugated with ubiquitin. In HEK293 cells, Nedd4 overexpression induced IRS-2 ubiquitination, which was decreased by USP15 co-expression while increased by USP15 knockdown. Nedd4 overexpression enhanced IGF-I-dependent IRS-2 tyrosine phosphorylation, and USP15 co-expression suppressed it. Conversely, USP15 knockdown increased IRS-2 tyrosine phosphorylation and downstream signaling in prostate cancer PC-3 cells. We concluded that USP15 attenuates IGF-I signaling by antagonizing Nedd4-induced IRS-2 ubiquitination.

DOI： 10.1016/j.bbrc.2017.01.101

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Insulin receptor substrates (IRSs) are well known to play crucial roles in mediating intracellular signals of insulin-like growth factors (IGFs)/insulin. Previously, we showed that IRS-1 forms high molecular mass complexes containing RNAs. To identify RNAs in IRS-1 complexes, we performed ultraviolet (UV) cross-linking and immunoprecipitation analysis using HEK293 cells expressing FLAG-IRS-1 and FLAG-IRS-2. We detected the radioactive signals in the immunoprecipitates of FLAG-IRS-1 proportional to the UV irradiation, but not in the immunoprecipitates of FLAG-IRS-2, suggesting the direct contact of RNAs with IRS-1. RNAs cross-linked to IRS-1 were then amplified by RT-PCR, followed by sequence analysis. We isolated sequence tags attributed to 25 messenger RNAs and 8 non-coding RNAs, including small nucleolar RNAs (snoRNAs). We focused on the interaction of IRS-1 with U96A snoRNA (U96A) and its host Rack1 (receptor for activated C kinase 1) pre-mRNA. We confirmed the interaction of IRS-1 with U96A, and with RACK1 pre-mRNA by immunoprecipitation with IRS-1 followed by Northern blotting or RT-PCR analyses. Mature U96A in IRS-1(-/-) mouse embryonic fibroblasts was quantitatively less than WT. We also found that a part of nuclear IRS-1 is localized in the Cajal body, a nuclear subcompartment where snoRNA mature. The unanticipated function of IRS-1 in snoRNA biogenesis highlights the potential of RNA-associated IRS-1 complex to open a new line of investigation to dissect the novel mechanisms regulating IGFs/insulin-mediated biological events.

DOI： 10.3389/fendo.2014.00024

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Language：Japanese   Publishing type：Research paper (conference, symposium, etc.)  

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OBJECTIVE: Hyperuricemia is common in patients with metabolic syndrome. We investigated the role of xanthine oxidoreductase (XOR) in atherosclerosis development, and the effects of the XOR inhibitor allopurinol on this process. METHODS AND RESULTS: Oral administration of allopurinol to ApoE knockout mice markedly ameliorated lipid accumulation and calcification in the aorta and aortic root. In addition, allopurinol treatment or siRNA-mediated gene knockdown of XOR suppressed transformation of J774.1 murine macrophage cells, treated with acetylated LDL or very low density lipoprotein (VLDL) into foam cells. This inhibitory effect of allopurinol was also observed in primary cultured human macrophages. In contrast, overexpression of XOR promoted transformation of J774.1 cells into foam cells. Interestingly, SR-A1, SR-B1, SR-B II, and VLDL receptors in J774.1 cells were reduced by XOR knockdown, and increased by XOR overexpression. Conversely, expressions of ABCA1 and ABCG1 were increased by XOR knockdown and suppressed by XOR overexpression. Finally, productions of inflammatory cytokines accompanied by foam cell formation were also reduced by allopurinol administration. CONCLUSIONS: These results strongly suggest XOR activity and/or its expression level to contribute to macrophage foam cell formation. Thus, XOR inhibitors may be useful for preventing atherosclerosis.

DOI： 10.1161/ATVBAHA.111.234559

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Language：Japanese   Publishing type：Research paper (conference, symposium, etc.)  

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Several lines of evidence have suggested a role of gut microbiota in the etiology of nonalcoholic steatohepatitis (NASH). NASH subjects reportedly showed a prolonged orocecal transit time coexistent with small intestinal bacterial overgrowth. We considered the possibility that enhanced gastrointestinal motility would influence gut microbiota and thus investigated the effects of the gastroprokinetic agent mosapride citrate (MC) on gut microbiota and the development of NASH using a methionine-choline deficient (MCD) diet-fed rodent model. Mice were divided into three groups, given the normal chow diet (NCD), the MCD diet, or the MCD diet containing 10 mg·kg(-1)·day(-1) of MC (MCD plus MC) for 6 wk. NASH development was evaluated based on hepatic histochemical findings, serum parameters and various mRNA and/or protein expression levels. MC treatment suppressed MCD diet-induced NASH development, with reduced serum lipopolysaccharide and increased plasma glucagon-like peptide-1 (GLP-1) concentrations. Calculation of the relative abundance of each strain based on gut microbiota analyses indicated lactic acid bacteria specifically, such as Bifidobacterium and Lactobacillus, in feces to be decreased in the MCD, compared with the NCD group. Interestingly, the reduction in lactic acid bacteria in the MCD diet group was reversed in the MCD plus MC group. In addition, colon inflammation observed in the MCD diet group was reduced in the MCD plus MC group. Therefore, MC showed a protective effect against MCD diet-induced NASH development in our rodent model, with possible involvements of increased fecal lactic acid bacteria, protection against colon inflammation and elevated plasma GLP-1.

DOI： 10.1152/ajpgi.00198.2014

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Insulin receptor substrates (IRSs) are phosphorylated by activated insulin/insulin-like growth factor (IGF)-I receptor tyrosine kinases. Phosphotyrosyl IRSs are recognized by signaling molecules possessing src homology region 2 (SH2) domains, which mediate various insulin/IGF bioactivities. However, we have shown that IRSs are also associated with other proteins by a phosphotyrosine-independent mechanism. Here, we demonstrated that IRSs form high-molecular-mass complexes (we named these complexes IRSomes) with various proteins and we elucidated their possible roles. Blue native-polyacrylamide gel electrophoresis of cell lysates revealed IRSome formation. Some proteins associated with IRSs in IRS-isoform-, cell-type-, or stimulus-specific manners. Results of the in vitro tyrosine phosphorylation assay indicated that tyrosine phosphorylation of IRS-1 by insulin receptor was decreased when IRS-1 was contained in IRSomes prepared from 3T3-L1 adipocytes treated with TNF-α. Also, tyrosine phosphorylation of IRS-2 by IGF-I receptor was increased when IRS-2 was contained in IRSomes prepared from FRTL-5 thyrocytes treated with dibutyryl cAMP. These results demonstrated that cytokine/hormone-induced formation of IRSomes modulates availability of IRSs to receptor tyrosine kinases.

DOI： 10.1016/j.bbrc.2010.12.045

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DOI： 10.1101/2023.12.27.573405

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The molecular evolution processes underlying the acquisition of the placenta in eutherian ancestors are not fully understood. Mouse NCK-interacting kinase (NIK)-related kinase (NRK) is expressed highly in the placenta and plays a role in preventing placental hyperplasia. Here, we show the molecular evolution of NRK, which confers its function for inhibiting placental cell proliferation. Comparative genome analysis identified NRK orthologs across vertebrates, which share the kinase and citron homology (CNH) domains. Evolutionary analysis revealed that NRK underwent extensive amino acid substitutions in the ancestor of placental mammals and has been since conserved. Biochemical analysis of mouse NRK revealed that the CNH domain binds to phospholipids, and a region in NRK binds to and inhibits casein kinase-2 (CK2), which we named the CK2-inhibitory region (CIR). Cell culture experiments suggest the following: 1) Mouse NRK is localized at the plasma membrane via the CNH domain, where the CIR inhibits CK2. 2) This mitigates CK2-dependent phosphorylation and inhibition of PTEN and 3) leads to the inhibition of AKT signaling and cell proliferation. Nrk deficiency increased phosphorylation levels of PTEN and AKT in mouse placenta, supporting our hypothesis. Unlike mouse NRK, chicken NRK did not bind to phospholipids and CK2, decrease phosphorylation of AKT, or inhibit cell proliferation. Both the CNH domain and CIR have evolved under purifying selection in placental mammals. Taken together, our study suggests that placental mammals acquired the phospholipid-binding CNH domain and CIR in NRK for regulating the CK2-PTEN-AKT pathway and placental cell proliferation.

DOI： 10.1093/molbev/msab371

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Ubiquitin-specific protease 8 (USP8) is a deubiquitinating enzyme involved in multiple membrane trafficking pathways. The enzyme activity is inhibited by binding to 14-3-3 proteins. Mutations in the 14-3-3-binding motif in USP8 are related to Cushing's disease. However, the molecular basis of USP8 activity regulation remains unclear. This study identified amino acids 645-684 of USP8 as an autoinhibitory region, which might interact with the catalytic USP domain, as per the results of pull-down and single-molecule FRET assays performed in this study. In silico modelling indicated that the region forms a WW-like domain structure, plugs the catalytic cleft, and narrows the entrance to the ubiquitin-binding pocket. Furthermore, 14-3-3 inhibited USP8 activity partly by enhancing the interaction between the WW-like and USP domains. These findings provide the molecular basis of USP8 autoinhibition via the WW-like domain. Moreover, they suggest that the release of autoinhibition may underlie Cushing's disease due to USP8 mutations.

DOI： 10.1038/s42003-021-02802-x

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We study a patient with the human papilloma virus (HPV)-2-driven "tree-man" phenotype and two relatives with unusually severe HPV4-driven warts. The giant horns form an HPV-2-driven multifocal benign epithelial tumor overexpressing viral oncogenes in the epidermis basal layer. The patients are unexpectedly homozygous for a private CD28 variant. They have no detectable CD28 on their T cells, with the exception of a small contingent of revertant memory CD4+ T cells. T cell development is barely affected, and T cells respond to CD3 and CD2, but not CD28, costimulation. Although the patients do not display HPV-2- and HPV-4-reactive CD4+ T cells in vitro, they make antibodies specific for both viruses in vivo. CD28-deficient mice are susceptible to cutaneous infections with the mouse papillomavirus MmuPV1. The control of HPV-2 and HPV-4 in keratinocytes is dependent on the T cell CD28 co-activation pathway. Surprisingly, human CD28-dependent T cell responses are largely redundant for protective immunity.

DOI： 10.1016/j.cell.2021.06.004

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Adrenal chromaffin cells and sympathetic neurons synthesize and release catecholamines, and both cell types are derived from neural crest precursors. However, they have different developmental histories, with sympathetic neurons derived directly from neural crest precursors while adrenal chromaffin cells arise from neural crest-derived cells that express Schwann cell markers. We have sought to identify the genes, including imprinted genes, which regulate the development of the two cell types in mice. We developed a method of separating the two cell types as early as E12.5, using differences in expression of enhanced yellow fluorescent protein driven from the tyrosine hydroxylase gene, and then used RNA sequencing to confirm the characteristic molecular signatures of the two cell types. We identified genes differentially expressed by adrenal chromaffin cells and sympathetic neurons. Deletion of a gene highly expressed by adrenal chromaffin cells, NIK-related kinase, a gene on the X-chromosome, results in reduced expression of adrenaline-synthesizing enzyme, phenyl-N-methyl transferase, by adrenal chromaffin cells and changes in cell cycle dynamics. Finally, many imprinted genes are up-regulated in chromaffin cells and may play key roles in their development.

DOI： 10.1038/s41598-019-40501-0

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Endocytic trafficking is regulated by ubiquitylation (also known as ubiquitination) of cargoes and endocytic machineries. The role of ubiquitylation in lysosomal delivery has been well documented, but its role in the recycling pathway is largely unknown. Here, we report that the ubiquitin (Ub) ligase RFFL regulates ubiquitylation of endocytic recycling regulators. An RFFL dominant-negative (DN) mutant induced clustering of endocytic recycling compartments (ERCs) and delayed endocytic cargo recycling without affecting lysosomal traffic. A BioID RFFL interactome analysis revealed that RFFL interacts with the Rab11 effectors EHD1, MICALL1 and class I Rab11-FIPs. The RFFL DN mutant strongly captured these Rab11 effectors and inhibited their ubiquitylation. The prolonged interaction of RFFL with Rab11 effectors was sufficient to induce the clustered ERC phenotype and to delay cargo recycling. RFFL directly ubiquitylates these Rab11 effectors in vitro, but RFFL knockout (KO) only reduced the ubiquitylation of Rab11-FIP1. RFFL KO had a minimal effect on the ubiquitylation of EHD1, MICALL1, and Rab11-FIP2, and failed to delay transferrin recycling. These results suggest that multiple Ub ligases including RFFL regulate the ubiquitylation of Rab11 effectors, determining the integral function of the ERC.

DOI： 10.1242/jcs.228007

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Insulin-like growth factors (IGFs) have been shown to induce proliferation of many types of cells. Insulin receptor substrates (IRSs) are major targets of IGF-I receptor (IGF-IR) tyrosine kinase activated by IGFs, and are known to play important roles in the activation of downstream signaling pathways, such as the Erk1/2 pathway. Dysregulation of IGF signaling represents a central tumor promoting principle in human carcinogenesis. Prostate carcinoma is highly dependent on the IGF/IGF-IR/IRS axis. Here we identified the deubiquitinase, ubiquitin specific peptidase 9X (USP9X) as a novel binding partner of IRS-2. In a human prostate carcinoma cell line, small interfering RNA (siRNA)-mediated knockdown of USP9X reduced IGF-IR as well as IRS-2 protein levels and increased their ubiquitination. Knockdown of USP9X suppressed basal activation of the Erk1/2 pathway, which was significantly restored by exogenous expression of IRS-2 but not by IGF-IR, suggesting that the stabilization of IRS-2 by USP9X is critical for basal Erk1/2 activation. Finally, we measured anchorage-independent cell growth, a characteristic cancer feature, by soft-agar colony formation assay. Knockdown of USP9X significantly reduced anchorage-independent cell growth of prostate carcinoma cell line. Taken all together, our findings indicate that USP9X is required for the promotion of prostate cancer growth by maintaining the activation of the Erk1/2 pathway through IRS-2 stabilization.

DOI： 10.18632/oncotarget.26049

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Stress granules are transient cytoplasmic foci induced by various stresses that contain translation-stalled mRNAs and RNA-binding proteins. They are proposed to modulate mRNA translation and stress responses. Here, we show that the deubiquitylases USP5 and USP13 are recruited to heat-induced stress granules. Heat-induced stress granules also contained K48- and K63-linked ubiquitin chains. Depletion of USP5 or USP13 resulted in elevated ubiquitin chain levels and accelerated assembly of heat-induced stress granules, suggesting that these enzymes regulate the stability of the stress granules through their ubiquitin isopeptidase activity. Moreover, disassembly of heat-induced stress granules after returning the cells to normal temperatures was markedly repressed by individual depletion of USP5 or USP13. Finally, overexpression of a ubiquitin mutant lacking the C-terminal diglycine motif caused the accumulation of unanchored ubiquitin chains and the repression of the disassembly of heat-induced stress granules. As unanchored ubiquitin chains are preferred substrates for USP5, we suggest that USP5 regulates the assembly and disassembly of heat-induced stress granules by mediating the hydrolysis of unanchored ubiquitin chains while USP13 regulates stress granules through deubiquitylating protein-conjugated ubiquitin chains.This article has an associated First Person interview with the first author of the paper.

DOI： 10.1242/jcs.210856

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Linear ubiquitin chain assembly complex (LUBAC), composed of SHARPIN (SHANK-associated RH domain-interacting protein), HOIL-1L (longer isoform of heme-oxidized iron-regulatory protein 2 ubiquitin ligase-1), and HOIP (HOIL-1L interacting protein), forms linear ubiquitin on nuclear factor-κB (NF-κB) essential modulator (NEMO) and induces NF-κB pathway activation. SHARPIN expression and LUBAC formation were significantly reduced in the livers of mice 24 h after the injection of either carbon tetrachloride (CCl₄) or acetaminophen (APAP), both of which produced the fulminant hepatitis phenotype. To elucidate its pathological significance, hepatic SHARPIN expression was suppressed in mice by injecting shRNA adenovirus via the tail vein. Seven days after this transduction, without additional inflammatory stimuli, substantial inflammation and fibrosis with enhanced hepatocyte apoptosis occurred in the livers. A similar but more severe phenotype was observed with suppression of HOIP, which is responsible for the E3 ligase activity of LUBAC. Furthermore, in good agreement with these in vivo results, transduction of Hepa1-6 hepatoma cells with SHARPIN, HOIL-1L, or HOIP shRNA adenovirus induced apoptosis of these cells in response to tumor necrosis factor-α (TNFα) stimulation. Thus, LUBAC is essential for the survival of hepatocytes, and it is likely that reduction of LUBAC is a factor promoting hepatocyte death in addition to the direct effect of drug toxicity.

DOI： 10.3390/ijms18020326

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Resistin-like molecule β (RELMβ) reportedly has multiple functions including local immune responses in the gut. In this study, we investigated the possible contribution of RELMβ to non-alcoholic steatohepatitis (NASH) development. First, RELMβ knock-out (KO) mice were shown to be resistant to methionine-choline deficient (MCD) diet-induced NASH development. Since it was newly revealed that Kupffer cells in the liver express RELMβ and that RELMβ expression levels in the colon and the numbers of RELMβ-positive Kupffer cells were both increased in this model, we carried out further experiments using radiation chimeras between wild-type and RELMβ-KO mice to distinguish between the contributions of RELMβ in these two organs. These experiments revealed the requirement of RELMβ in both organs for full manifestation of NASH, while deletion of each one alone attenuated the development of NASH with reduced serum lipopolysaccharide (LPS) levels. The higher proportion of lactic acid bacteria in the gut microbiota of RELMβ-KO than in that of wild-type mice may be one of the mechanisms underlying the lower serum LPS level the former. These data suggest the contribution of increases in RELMβ in the gut and Kupffer cells to NASH development, raising the possibility of RELMβ being a novel therapeutic target for NASH.

DOI： 10.1038/srep20157

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DOI： 10.1016/bs.pmbts.2016.04.001

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Language：English   Publisher：(公社)日本生化学会  

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Dipeptidyl peptidase IV (DPP-IV) expression in visceral adipose tissue is reportedly increased in obese patients, suggesting an association of DPP-IV with inflammation. In this study, first, lipopolysaccharide (LPS)- or palmitate-induced elevations of inflammatory cytokine mRNA expressions in RAW264.7 macrophages were shown to be significantly suppressed by coincubation with a DPP-IV inhibitor, anagliptin (10 μM), despite low DPP-IV expression in the RAW264.7 cells. Regarding the molecular mechanism, LPS-induced degradation of IκBα and phosphorylations of p65, JNK, and p38, as well as NF-κB and AP-1 promoter activities, were revealed to be suppressed by incubation with anagliptin, indicating suppressive effects of anagliptin on both NF-κB and AP-1 signaling pathways. Anagliptin also acted on 3T3-L1 adipocytes, weakly suppressing the inflammatory cytokine expressions induced by LPS and TNFα. When 3T3-L1 and RAW cells were cocultured and stimulated with LPS, the effects of anagliptin on the suppression of cytokine expressions in 3T3-L1 adipocytes were more marked and became evident at the 10 μM concentration. Anti-inflammatory effects of anagliptin were also observed in vivo on the elevated hepatic and adipose expressions and serum concentrations of inflammatory cytokines in association with the suppression of hepatic NF-κB transcriptional activity in LPS-infused mice. Taking these observations together, the anti-inflammatory properties of anagliptin may be beneficial in terms of preventing exacerbation of diabetes and cardiovascular events.

DOI： 10.1152/ajpendo.00553.2014

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Xanthine oxidase (XO) is an enzyme involved in the production of uric acid (UA) from purine nucleotides. Numerous recent studies have revealed the likelihood of metabolic syndrome including nonalcoholic fatty liver disease (NAFLD) or steatohepatitis (NASH) to be related to hyperuricemia. However, it remains unclear whether elevated serum UA during the development of NAFLD or NASH is a cause or a consequence of these diseases. In this study, the XO inhibitor febuxostat was administered to two types of NASH model mice. Febuxostat exerted a strong protective effect against NASH development induced by a high-fat diet containing trans fatty acid (HFDT). In contrast, methionine choline-deficient-diet-induced NASH development not accompanied by hyperuricemia showed no UA normalization, suggesting that the ameliorating effect of febuxostat occurs via the normalization of hyperuricemia itself and/or accompanying molecular mechanism(s) such as oxidative stress. In the HFDT-fed mice, hyperuricemia, elevated alanine aminotransferase, and increased Tunnel-positive cells in the liver were normalized by febuxostat administration. In addition, upregulation of fatty acid oxidation-related genes, fibrotic change, and increases in collagen deposition, inflammatory cytokine expressions, and lipid peroxidation in the HFDT-fed mice were also normalized by febuxostat administration. Taken together, these observations indicate that administration of febuxostat has a protective effect against HFDT-induced NASH development, suggesting the importance of XO in its pathogenesis. Thus XO inhibitors are potentially potent therapies for patients with NASH, particularly that associated with hyperuricemia.

DOI： 10.1152/ajpgi.00443.2014

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BACKGROUND: Prostate cancer initially develops in an androgen-dependent manner but, during its progression, transitions to being androgen-independent in the advanced stage. Pin1, one of the peptidyl-prolyl cis/trans isomerases, is reportedly overexpressed in prostate cancers and is considered to contribute to accelerated cell growth, which may be one of the major factors contributing to their androgen-independent growth. Thus, we investigated how Pin1 modulates the gene expressions in both androgen-dependent and androgen-independent prostate cancer cell lines using microarray analysis. In addition, the effects of Juglone, a commercially available Pin1 inhibitor were also examined. METHODS: Two prostate cancer cell-lines, LNCaP (androgen-dependent) and DU145 (androgen-independent), were treated with Pin1 siRNA and its effects on gene expressions were analyzed by microarray. Individual gene regulations induced by Pin1 siRNA or the Pin1 inhibitor Juglone were examined using RT-PCR. In addition, the effects of Juglone on the growth of LNCaP and DU145 transplanted into mice were investigated. RESULTS: Microarray analysis revealed that transcriptional factors regulated by Pin1 differed markedly between LNCaP and DU145 cells, the only exception being that Nrf was regulated in the same way by Pin1 siRNA in both cell lines. Despite this marked difference in gene regulations, Pin1 siRNA and Juglone exert a strong inhibitory effect on both the LNCaP and the DU145 cell line, suppressing in vitro cell proliferation as well as tumor enlargement when transplanted into mice. CONCLUSIONS: Despite Pin1-regulated gene expressions differing between these two prostate cancer cell-lines, LNCaP (androgen-dependent) and DU145 (androgen-independent), Pin1 inhibition suppresses proliferation of both cell-lines. These findings suggest the potential effectiveness of Pin1 inhibitors as therapeutic agents for prostate cancers, regardless of their androgen sensitivity.

DOI： 10.1371/journal.pone.0127467

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Nonalcoholic steatohepatitis (NASH) is a disorder characterized by hepatic lipid accumulation followed by the inflammation-induced death of hepatocytes and fibrosis. In this process, oxidative stress contributes to the induction of several inflammatory cytokines including TNF-α andIL-1β in macrophages, while, in hepatocytes, NF-κB reportedly induces the expressions of cell survival genes for protection from apoptosis. Recently, it was reported that the new ubiquitin ligase complex termed linear ubiquitin chain assembly complex (LUBAC), composed of SHARPIN (SHANK-associated RH domain-interacting protein), HOIL-1L (longer isoform of heme-oxidized iron-regulatory protein 2 ubiquitin ligase-1), and HOIP (HOIL-1L interacting protein), forms linear ubiquitin on NF-κB essential modulator (NEMO) and thereby induces NF-κB pathway activation. In this study, we demonstrated the formation of LUBAC to be impaired in the livers of NASH rodent models produced by methionine and choline deficient (MCD) diet feeding, first by either gel filtration or Blue Native-PAGE, with subsequent confirmation by western blotting. The reduction of LUBAC is likely to be attributable to markedly reduced expression of SHARPIN, one of its components. Thus, impaired LUBAC formation, which would result in insufficient NF-κB activation, may be one of the molecular mechanisms underlying the enhanced apoptotic response of hepatocytes in MCD diet-induced NASH livers.

DOI： 10.1155/2015/125380

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Insulin-like peptides, such as insulin-like growth factors (IGFs) and insulin, induce a variety of bioactivities, such as growth, differentiation, survival, increased anabolism, and decreased catabolism in many cell types and in vivo. In general, IGFs or insulin bind to IGF-I receptor (IGF-IR) or insulin receptor (IR), activating the receptor tyrosine kinase. Insulin receptor substrates (IRSs) are known to be major substrates of receptor kinases, mediating IGF/insulin signals to direct bioactivities. Recently, we discovered that IRSs form high-molecular-mass complexes (referred to here as IRSomes) even without IGF/insulin stimulation. These complexes contain proteins (referred to here as IRSAPs; IRS-associated proteins), which modulate tyrosine phosphorylation of IRSs by receptor kinases, control IRS stability, and determine intracellular localization of IRSs. In addition, in these complexes, we found not only proteins that are involved in RNA metabolism but also RNAs themselves. Thus, IRSAPs possibly contribute to modulation of IGF/insulin bioactivities. Since it is established that disorder of modulation of insulin-like activities causes various age-related diseases including cancer, we could propose that the IRSome is an important target for treatment of these diseases.

DOI： 10.3389/fendo.2015.00073

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Language：Japanese   Publisher：(一社)日本肥満学会  

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Inflammation involving adipose tissue is regarded as one of the major molecular mechanisms underlying obesity-related insulin resistance. Recent studies have suggested a series of angiotensin II receptor blockers (ARBs) to improve insulin resistance or protect against the development of diabetes mellitus. We previously demonstrated that valsartan suppresses the inflammatory response of macrophages. Interestingly, however, this effect did not occur via peroxisome proliferator-activated receptor (PPAR) γ or the AT1a receptor. This suppression appears to secondarily lead to amelioration of insulin resistance and reductions in abnormal gene expressions in adipocytes. In addition to these in vitro findings, we herein demonstrate the in vivo effects of valsartan, using mice constitutively infused with lipopolysaccharide (LPS) for 4 weeks. Oral administration of valsartan to LPS-infused mice normalized the increased expressions of inflammatory cytokines in adipose and liver tissues. These results raise the possibility that valsartan not only contributes to normalization of obesity-related insulin resistance, but is also beneficial for the treatment of other diseases with inflammation related to the metabolic syndrome such as atherosclerosis and non-alcoholic steatohepatitis. Further study is necessary to clarify these issues.

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Macrophages are integrated into adipose tissues and interact with adipocytes in obese subjects, thereby exacerbating adipose insulin resistance. This study aimed to elucidate the molecular mechanism underlying the insulin-sensitizing effect of the angiotensin II receptor blocker (ARB) valsartan, as demonstrated in clinical studies. Insulin signaling, i.e., insulin receptor substrate-1 and Akt phosphorylations, in 3T3-L1 adipocytes was impaired markedly by treatment with tumor necrosis factor-α (TNFα) or in the culture medium of lipopolysaccharide (LPS)-stimulated RAW 264.7 murine macrophages, and valsartan had no effects on these impairments. However, in contrast, when cocultured with RAW 264.7 cells using a transwell system, the LPS-induced insulin signaling impairment in 3T3-L1 adipocytes showed almost complete normalization with coaddition of valsartan. Furthermore, valsartan strongly suppressed LPS-induced productions of cytokines such as interleukin (IL)-1β, IL-6, and TNFα with nuclear factor-κB activation and c-Jun NH(2)-terminal kinase phosphorylation in RAW 264.7 and primary murine macrophages. Very interestingly, this effect of valsartan was also observed in THP-1 cells treated with angiotensin II type 1 (AT1) siRNA or a peroxisome proliferator-activated receptor-γ (PPARγ) antagonist as well as macrophages from AT1a receptor-knockout mice. We conclude that valsartan suppresses the inflammatory response of macrophages, albeit not via PPARγ or the AT1a receptor. This suppression appears to secondarily improve adipose insulin resistance.

DOI： 10.1152/ajpendo.00324.2011

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Language：Japanese   Publisher：(公社)日本小児科学会  

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CONTEXT: Mouse knockout models have clearly demonstrated the critical importance of IGF-I and IGF receptor type 1 (IGF-IR) for embryonic growth as well as postnatal growth. OBJECTIVE: We hypothesized that mutations of IGF-IR gene might predispose to short stature in children born with intrauterine growth retardation (IUGR). PATIENTS: Twenty-four children with unexplained IUGR (birth weight < -1.5 SD) and short stature (<-2.0 SD) were screened for abnormalities of the IGF-IR gene. METHODS: Direct DNA sequencing was used to identify IGF-IR gene mutations. Unprocessed IGF-IR proreceptor in fibroblasts was detected by immunoblot analysis. Functions of mutated IGF-IR in fibroblasts were evaluated by IGF-I binding, and IGF-I-stimulated DNA synthesis and beta-subunit autophosphorylation. RESULTS: We found the following results: 1) a heterozygous mutation (R709Q) changing the cleavage site from Arg-Lys-Arg-Arg to Arg-Lys-Gln-Arg was identified in a 6-yr-old Japanese girl (case 1) and her mother who also had IUGR with short stature (case 2); 2) fibroblasts from case 2 contained more IGF-IR proreceptor protein (189 +/- 26% of normal) and less mature beta-subunit protein (63 +/- 12%); 3) [125I]IGF-I binding to fibroblasts from case 2 was reduced, compared with normal control (0.61 +/- 0.16 x 10(6) vs. 1.14 +/- 0.12 x 10(6) sites per cell; P < 0.05); and 4) both IGF-I-stimulated [3H]thymidine incorporation and IGF-IR beta-subunit autophosphorylation were low in fibroblasts from case 2, compared with those of control (P < 0.05). CONCLUSIONS: These findings strongly suggest that this mutation leads to failure of processing of the IGF-IR proreceptor to mature IGF-IR and causes short stature and IUGR.

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The type 1 IGF receptor (IGF1R) is required for normal embryonic and postnatal growth. The aim of this study was to determine whether we could detect abnormal IGF1R function in skin fibroblasts from children with an abnormal copy number of the IGF1R gene. We report two children with altered copy number of the IGF1R gene who presented with abnormal growth. Case 1 is a girl with intrauterine growth retardation, postnatal growth failure, and recurrent hypoglycemia. Pituitary function tests were normal. Routine karyotype analysis identified a deletion on 15q26.2, and a fluorescence in situ hybridization study using IGF1R probes showed only a single IGF1R gene. Case 2 was large for gestational age, with birth weight and length at or above 97th percentile, and showed rapid early postnatal growth. He was found to have a recombinant chromosome 15 containing a partial duplication at 15q (q25-qter). A fluorescence in situ hybridization study using the same probes showed three copies of the IGF1R gene. In a mitochondrial activity assay, skin fibroblasts from the subject with only one copy of IGF1R showed slower growth, whereas cells from the subject with three copies of IGF1R showed accelerated growth compared with controls. IGF1R phosphorylation, as assessed by Western blot, and IGF1R binding studies were decreased compared with controls in the child with one copy of the IGF1R and increased in the child with three copies of the gene. Our data are consistent with the concept that IGF1R gene copy number is of functional and clinical importance in humans.

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DOI： 10.1016/S0167-4889(01)00119-7

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Language：English   Publishing type：Research paper, summary (international conference)  

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Language：Japanese   Publisher：(一社)日本内分泌学会  

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Language：Japanese   Publisher：(一社)日本糖尿病学会  

J-GLOBAL

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Language：Japanese   Publisher：(一社)日本糖尿病学会  

J-GLOBAL

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Language：Japanese   Publisher：(一社)日本糖尿病学会  

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J-GLOBAL

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Language：English   Publishing type：Research paper, summary (international conference)  

Web of Science

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Language：Japanese   Publisher：(一社)日本糖尿病学会  

J-GLOBAL

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Language：Japanese   Publisher：(一社)日本糖尿病学会  

J-GLOBAL

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Language：Japanese   Publisher：(一社)日本糖尿病学会  

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J-GLOBAL

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Language：Japanese   Publisher：(公社)日本生化学会  

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Language：Japanese   Publisher：(一社)日本糖尿病学会  

J-GLOBAL

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Language：Japanese   Publisher：(一社)日本糖尿病学会  

J-GLOBAL

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Language：Japanese   Publisher：(一社)日本糖尿病学会  

J-GLOBAL

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Language：Japanese   Publisher：(一社)日本糖尿病学会  

J-GLOBAL

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J-GLOBAL

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Language：Japanese   Publisher：(公社)日本生化学会  

J-GLOBAL

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Language：Japanese   Publisher：(公社)日本生化学会  

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Language：Japanese   Publisher：(公社)日本生化学会  

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Language：Japanese   Publisher：(公社)日本生化学会  

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Language：English   Publishing type：Research paper, summary (international conference)  

Web of Science

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Language：Japanese  

CiNii Books

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Other Link： http://search.jamas.or.jp/link/ui/2009337890

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Language：Japanese  

CiNii Books

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Language：Japanese   Presentation type：Poster presentation  

Venue：横浜  

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Language：Japanese   Presentation type：Poster presentation  

Venue：横浜  

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Language：Japanese   Presentation type：Poster presentation  

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Language：Japanese   Presentation type：Oral presentation (general)  

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Language：English   Presentation type：Poster presentation  

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Language：Japanese   Presentation type：Oral presentation (general)  

Venue：松山  

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Language：Japanese   Presentation type：Oral presentation (general)  

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Language：English   Presentation type：Poster presentation  

Venue：Cavtat  

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Language：Japanese   Presentation type：Poster presentation  

Venue：神戸  

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Language：Japanese   Presentation type：Poster presentation  

Venue：神戸  

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Language：English   Presentation type：Poster presentation  

Venue：神戸  

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Language：English   Presentation type：Poster presentation  

Venue：東京都  

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Language：English   Presentation type：Oral presentation (general)  

Venue：東京都  

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Language：English   Presentation type：Oral presentation (general)  

Venue：東京都  

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Language：Japanese   Presentation type：Poster presentation  

Venue：千葉県  

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Language：Japanese   Presentation type：Poster presentation  

Venue：名古屋  

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Language：Japanese   Presentation type：Poster presentation  

Venue：神戸  

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Language：Japanese   Presentation type：Poster presentation  

Venue：神戸  

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Language：Japanese   Presentation type：Poster presentation  

Venue：神戸  

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Language：Japanese   Presentation type：Poster presentation  

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Language：English   Presentation type：Oral presentation (general)  

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Language：English   Presentation type：Poster presentation  

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Language：English   Presentation type：Poster presentation  

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Language：English   Presentation type：Poster presentation  

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Language：Japanese   Presentation type：Oral presentation (general)  

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Language：English   Presentation type：Oral presentation (invited, special)  

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Language：Japanese   Presentation type：Oral presentation (general)  

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Language：English   Presentation type：Poster presentation  

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Language：English   Presentation type：Poster presentation  

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Language：English   Presentation type：Oral presentation (general)  

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Language：English   Presentation type：Poster presentation  

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Language：Japanese   Presentation type：Poster presentation  

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Language：English   Presentation type：Oral presentation (general)  

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Language：Japanese   Presentation type：Oral presentation (general)  

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Language：English   Presentation type：Oral presentation (invited, special)  

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Language：English   Presentation type：Oral presentation (general)  

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Language：English   Presentation type：Poster presentation  

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Language：English   Presentation type：Poster presentation  

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Language：English   Presentation type：Poster presentation  

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