Shotaro Nakajima

University of Yamanashi, Kōhu, Yamanashi, Japan

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Publications (26)110.66 Total impact

  • Shotaro Nakajima · Yuan Chi · Kun Gao · Koji Kono · Jian Yao ·
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    ABSTRACT: Salubrinal is a selective inhibitor of cellular complexes that dephosphorylate eukaryotic translation initiation factor 2α (eIF2α). In previous reports, salubrinal was shown to have the potential to inhibit the activation of nuclear factor-κB (NF-κB) by several stimuli. However, the effects of salubrinal on NF-κB signaling are largely unknown. In this study, we investigated whether and how salubrinal affects NF-κB activation induced by tumor necrosis factor (TNF)-α and interleukin (IL)-1β. We found that salubrinal selectively blocked TNF-α- but not IL-1β-induced activation of NF-κB. This inhibitory effect occurred upstream of transforming growth factor (TGF)-β-activated kinase 1 (TAK1). Further experiments revealed that salubrinal blocked TNF-α-triggered NF-κB activation independent of its action on eIF2α because knockdown of eIF2α by small interfering RNA (siRNA) did not reverse the inhibitory effect of salubrinal on NF-κB. Moreover, guanabenz, a selective inhibitor of the regulatory subunit of protein phosphatase (PP) 1, also preferentially inhibited TNF-α-triggered activation of NF-κB. These findings raise the possibility that salubrinal may selectively block TNF-α-triggered activation of the NF-κB pathway through inhibition of the PP1 complex.
    Biological & Pharmaceutical Bulletin 09/2015; 38(9):1368-74. DOI:10.1248/bpb.b15-00312 · 1.83 Impact Factor
  • Yuan Chi · Kun Gao · Kai Li · Shotaro Nakajima · Satoru Kira · Masayuki Takeda · Jian Yao ·
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    ABSTRACT: Connexin hemichannels regulate many cell functions. However, the molecular mechanisms involved remain elusive. Given that hemichannel opening causes loss of ATP, we therefore speculated a potential implication of AMPK in the biological actions of hemichannels. Activation of hemichannels by removing extracellular Ca(2+) led to an efflux of ATP and a weak activation of AMPK. Unexpectedly, dysfunction of hemichannels markedly potentiated AMPK activation, which was reproduced by promotion of extracellular ATP degradation or inhibition of P2 purinoceptors, but counteracted by exogenous ATP. Further analysis revealed that ATP induced a purinoceptor-dependent activation of Akt and mTOR. Suppression of Akt or mTOR augmented AMPK activation, whereas activation of Akt by transfection of cells with myr-Akt, a constitutively active form of Akt, abolished AMPK activation. In a pathological model of hemichannel opening triggered by cadmium, disclosure of hemichannels similarly enhanced AMPK activity, which protected cells from cadmium-induced cell injury through suppression of mTOR. Collectively, we unraveled a channel-mediated regulation of AMPK through purinergic signaling pathway. Furthermore, we defined AMPK as a pivotal molecule underlying the regulatory effects of hemichannels on cell survival.
    Journal of Cell Science 02/2014; 127(7). DOI:10.1242/jcs.139089 · 5.43 Impact Factor
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    ABSTRACT: 4-Phenylbutyric acid (4-PBA) is a chemical chaperone that eliminates accumulation of unfolded proteins in the endoplasmic reticulum (ER). However, its chaperoning potential is often weak and insufficient to attenuate the unfolded protein response (UPR) in vitro and in vivo. Towards development of more potent chemical chaperones, we synthesized six analogues of 4-PBA, and their pharmacological actions on the UPR were evaluated. NRK-52E cells were treated with ER stress inducers (tunicamycin or thapsigargin) in the presence of individual 4-PBA analogues, and their suppressive effects on the UPR were assessed using selective indicators for individual UPR pathways. 2-POAA-OMe, 2-POAA-NO2 and 2-NOAA, but not others, suppressed induction of ER stress markers GRP78 and CHOP. This suppressive effect was more potent than that of 4-PBA. Among three major UPR branches, the IRE1 and ATF6 pathways were markedly blocked by these compounds, which was evidenced by suppression of XBP1 splicing, inhibition of UPRE and ERSE activation and inhibition of JNK phosphorylation. Unexpectedly, however, these agents did not inhibit phosphorylation of PERK and eIF2α triggered by ER stress. In ER stress-exposed cells, early activation of NF-κB was inhibited by these compounds dose-dependently. 2-POAA-OMe and 2-POAA-NO2 also inhibited ER stress-induced phosphorylation of Akt. We developed 4-PBA analogues that strongly inhibit activation of the IRE1 and ATF6 pathways and downstream pathogenic targets including NF-κB and Akt in ER stress-exposed cells. These compounds would be useful for efficient therapeutic intervention in ER stress-related pathological processes.
    British Journal of Pharmacology 07/2013; 170(4). DOI:10.1111/bph.12306 · 4.84 Impact Factor
  • Shotaro Nakajima · Masanori Kitamura ·
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    ABSTRACT: Nuclear factor-κB (NF-κB) is a transcription factor that plays a crucial role in coordinating innate and adaptive immunity, inflammation and apoptotic cell death. NF-κB is activated by various inflammatory stimuli including peptide factors and infectious microbes. It is also known as a redox-sensitive transcription factor activated by reactive oxygen species (ROS). Over the past decades, various investigators focused onthe role of ROS in the activation of NF-κB by cytokines and lipopolysaccharide. However, recent studies also suggested that ROS have the potential to repress NF-κB activity. Currently, it is not well addressed how ROS regulate activity of NF-κB in a bidirectional fashion. In this article, we summarize evidence for positive and negative regulation of NF-κB by ROS, possible redox-sensitive targets for NF-κB signaling and mechanisms underlying biphasic and bidirectional influences of ROS on NF-κB, especially focusing on a role of ROS-mediated induction of endoplasmic reticulum stress.
    Free Radical Biology and Medicine 06/2013; 65. DOI:10.1016/j.freeradbiomed.2013.06.020 · 5.74 Impact Factor
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    ABSTRACT: In the present study, we show that cordycepin (3'-deoxyadenosine), a major nucleoside isolated from Cordyceps species, attenuates genotoxic stress-induced senescence. Etoposide- or doxorubicin-treated cells exhibited senescent morphology, growth arrest and positive staining for senescence-associated β-galactosidase. The induction of the senescent phenotype was inhibited by the treatment of cordycepin. This suppression was correlated with blunted activation of the p16(INK4a) and p21(WAF1/CIP1) gene promoters, as well as a decreased level of p21 (WAF1/CIP1) mRNA. Other adenosine-related substances including ATP, ADP and adenosine did not mimic the suppressive effect of cordycepin. The anti-senescence effect of cordycepin was mediated by activation of eukaryotic translation initiation factor 2α (eIF2α), because; 1) cordycepin induced phosphorylation of eIF2α, 2) selective activation of eIF2α mimicked the suppressive effect of cordycepin on senescence, and 3) functional knockdown of eIF2α reversed the effect of cordycepin. Unexpectedly, induction of p53 by etoposide was not inhibited by cordycepin, whereas 1) expression of Sp1 (required for the induction of p21(WAF1/CIP1) and activation of p16(INK4a) by genotoxic stress) was attenuated by cordycepin, 2) DNA binding activity of Sp1 was also inhibited, and 3) selective inhibition of Sp1 reproduced the suppressive effect of cordycepin on senescence. These results suggest that cordycepin interferes with senescence signaling via activation of eIF2α and suppression of Sp1 without affecting the level of p53.
    Toxicological Sciences 05/2013; 134(2). DOI:10.1093/toxsci/kft111 · 3.85 Impact Factor
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    ABSTRACT: Dehydroxymethylepoxyquinomicin (DHMEQ) is a low-m.w. compound that strongly inhibits NF-κB. Previous reports showed that DHMEQ directly binds to specific cysteine residues of NF-κB subunits and thereby inhibits their nuclear translocation and DNA binding. In this work, we describe novel mechanisms by which DHMEQ suppresses cytokine-triggered activation of NF-κB. We found that sustained exposure of renal tubular cells to DHMEQ blocked TNF-α- and IL-1β-induced TGF-β-activated kinase 1 (TAK1) phosphorylation, a crucial event for NF-κB activation upstream of IκB kinase. This inhibition was mediated by reactive oxygen species (ROS), because of the following: 1) DHMEQ caused generation of ROS; 2) pretreatment with ROS generator inhibited cytokine-induced TAK1 phosphorylation and NF-κB activation; and 3) scavenging of ROS attenuated the suppressive effects of DHMEQ on TAK1 and NF-κB. We also found that DHMEQ caused the unfolded protein response (UPR) through generation of ROS. Alleviation of the UPR by chemical and genetic chaperones partially attenuated the suppressive effect of DHMEQ on NF-κB. The UPR-mediated inhibition of NF-κB occurred downstream of degradation of IκBα and phosphorylation of p65. Subsequent experiments revealed the following: 1) DHMEQ caused selective induction of C/EBPβ through the UPR; 2) overexpression of C/EBPβ suppressed activation of NF-κB; 3) knockdown of C/EBPβ attenuated the inhibitory effect of DHMEQ; and 4) DHMEQ-induced expression of C/EBPβ did not affect TNF-α-triggered degradation of IκBα and phosphorylation of p65. These results suggest that, in addition to its known effect on nuclear translocation of NF-κB, DHMEQ interferes with the cytokine-induced NF-κB signaling via generation of ROS at both upstream and downstream of the IκB kinase-IκB level.
    The Journal of Immunology 05/2013; 190(12). DOI:10.4049/jimmunol.1300155 · 4.92 Impact Factor
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    ABSTRACT: Cordyceps militaris has been used in Eastern countries for the treatment of various diseases including chronic kidney diseases. However, there are no reports that identified its active entities and molecular mechanisms underlying its therapeutic effectiveness. 3'-Deoxyadenosine is a major nucleoside derivative isolated from C. militaris. Some reports suggested that both C. militaris and 3'-deoxyadenosine have anti-inflammatory and anti-fibrotic effects. In the present report, we investigated whether and how 3'-deoxyadenosine interferes with fibrogenic processes in the kidney. For this purpose, we examined effects of 3'-deoxyadenosine on the expression of collagens triggered by transforming growth factor-β (TGF-β1) and bone morphogenetic protein-4 (BMP-4), especially focusing on the regulation of Smad signaling in vitro and in vivo. We found that 3'-deoxyadenosine suppressed expression of collagens induced by TGF-β1 and BMP-4 dose dependently. This suppression occurred at the transcriptional level and was correlated with blunted activation of the CAGA box and the BMP-responsive element. The suppressive effect on the TGF-β/BMP signaling was mediated mainly by adenosine transporter and partially by the A3 adenosine receptor, but not A1/A2 adenosine receptors. 3'-Deoxyadenosine reduced levels of both phosphorylated and total Smad proteins (Smad1, 2 and 3) dose dependently. It was mainly ascribed to transcriptional suppression, but not to enhanced protein degradation and eIF2α-mediated translational suppression. Consistent with the in vitro results, in vivo administration with 3'-deoxyadenosine reduced the levels of phosphorylated and total Smad proteins, as well as the levels of Smad mRNAs, in the kidney subjected to unilateral ureteral obstruction. It was associated with blunted induction of type I collagen and α-smooth muscle actin, a decrease in the number of interstitial myofibroblasts and reduced fibrotic area. These results suggest that 3'-deoxyadenosine interferes with the TGF-β and BMP signaling via downregulation of Smads, which may underlie the anti-fibrotic effect of this agent. 3'-Deoxyadenosine may be useful for therapeutic intervention in various TGF-β-related fibrotic disorders.Laboratory Investigation advance online publication, 25 February 2013; doi:10.1038/labinvest.2013.4.
    Laboratory Investigation 02/2013; 93(4). DOI:10.1038/labinvest.2013.4 · 3.68 Impact Factor
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    ABSTRACT: During recovery from acute glomerulonephritis, cell proliferation, matrix expansion, and expression of the dedifferentiation marker α-smooth muscle actin (α-SMA) subside spontaneously. However, the molecular mechanisms underlying this recovery process remain elusive. In mesangioproliferative glomerulonephritis, the unfolded protein response (UPR) is induced in activated, dedifferentiated mesangial cells. We investigated the role of the UPR in mesangial cell deactivation and redifferentiation and found that, during experimental glomerulonephritis in rats, reinforcement of the UPR significantly attenuated mesangial cell proliferation, matrix expansion, and expression of α-SMA. Consistent with this in vivo result, induction of the UPR suppressed cell proliferation and transcriptional expression of type IV collagen (ColIV) and α-SMA in activated mesangial cells. The UPR reduced phosphorylation of Akt in vitro and in vivo, and it was responsible for attenuation of cell proliferation. The UPR also preferentially depressed levels of total and phosphorylated Smads without affecting transcriptional levels, and it was responsible for suppression of ColIV and α-SMA. Translational suppression via the eIF2α pathway, but not proteasome-mediated protein degradation, was responsible for the down-regulation of Smads. These results suggest the novel potential of the UPR to facilitate a phenotypic shift of activated glomerular cells toward deactivation and redifferentiation. The UPR may serve as endogenous machinery that supports recovery of glomeruli from acute inflammation.
    American Journal Of Pathology 09/2012; 181(6). DOI:10.1016/j.ajpath.2012.08.015 · 4.59 Impact Factor
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    M Kadomatsu · S Nakajima · H Kato · L Gu · Y Chi · J Yao · M Kitamura ·
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    ABSTRACT: Cordycepin (3'-deoxyadenosine) is one of the major bioactive substances produced by Cordyceps militaris, a traditional medicinal mushroom. Cordycepin possesses several biological activities, including both pro-apoptotic and anti-apoptotic properties. In the present report, we investigated an effect of cordycepin on the survival of cells exposed to tumour necrosis factor (TNF)-α. We found that subtoxic doses of cordycepin increased susceptibility of cells to TNF-α-induced apoptosis. It was associated with suppression of nuclear factor-κB (NF-κB), a major prosurvival component involved in TNF-α signalling. The adenosine transporter and A₃ adenosine receptor, but not A₁ and A₂ adenosine receptors, mediated both anti-NF-κB and pro-apoptotic effects. We found that cordycepin had the potential to phosphorylate eukaryotic translation initiation factor 2α (eIF2α) and that activation of eIF2α mimicked the suppressive effect of cordycepin on the NF-κB pathway. Furthermore, activation of eIF2α sensitized cells to TNF-α-induced apoptosis. To identify molecular events downstream of eIF2α, the role of mammalian target of rapamycin complex 1 (mTORC1) was examined. Selective activation of ₃eIF2α, as well as treatment with cordycepin, caused phosphorylation of mTORC1. Rapamycin, an inhibitor of mTORC1, significantly reversed the suppressive effects of eIF2α on NF-κB. These results suggest that cordycepin sensitizes cells to TNF-α-induced apoptosis, at least in part, via induction of the eIF2α-mTORC1 pathway and consequent suppression of NF-κB.
    Clinical & Experimental Immunology 06/2012; 168(3):325-32. DOI:10.1111/j.1365-2249.2012.04580.x · 3.04 Impact Factor
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    ABSTRACT: Background Bacterial peritonitis is a frequent complication in patients on peritoneal dialysis (PD). We previously reported that PD fluid (PDF) suppressed expression of monocyte chemoattractant protein 1 (MCP-1) in mesothelial cells in vitro and in vivo, which was ascribed to the suppression of nuclear factor-κB (NF-κB). To elucidate molecular mechanisms underlying this effect, we tested a role of endoplasmic reticulum (ER) stress. Methods Mesothelial cells and other cell types were exposed to acidic stress, and induction of the unfolded protein response was examined. Peritoneal induction of ER stress was also tested in mice exposed to acidic and neutralized PDF. Activation of NF-κB and expression of MCP-1 by tumour necrosis factor-α were evaluated in mesothelial cells under acidic and ER stress conditions. Peritoneal expression of MCP-1 and infiltration of monocytes were compared in lipopolysaccharide (LPS)-treated mice between normal and ER stress conditions.ResultsPDF, but not neutralized PDF, caused ER stress in the peritoneum. In vitro, acidic stress, but not metabolic and osmotic stress, induced ER stress in mesothelial cells and other cell types and suppressed activation of NF-κB and NF-κB-dependent MCP-1 induction. This effect was reproducible by other ER stress inducers, and attenuation of ER stress reversed the suppressive effect of low pH on NF-κB. Like PDF, ER stress inducers suppressed expression of MCP-1 and infiltration of mononuclear cells in the peritoneum of LPS-treated mice.Conclusion These results indicate a role for the acidic stress-ER stress pathway in blunted activation of NF-κB, which may cause perturbation of monocyte recruitment by mesothelial cells in PD patients. © The Author 2012. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved.
    Nephrology Dialysis Transplantation 05/2012; 27(11). DOI:10.1093/ndt/gfs130 · 3.58 Impact Factor
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    ABSTRACT: BACKGROUND AND PURPOSE Cordyceps militaris has the potential to suppress differentiation of pre-adipocytes. However, the active entities in the extract and the underlying mechanisms of its action are not known. Hence, we investigated whether and how cordycepin (3'-deoxyadenosine), a constituent of C. militaris, inhibits adipogenesis. EXPERIMENTAL APPROACH Differentiation of 3T3-L1 pre-adipocytes and pre-adipocytes in primary cultures was induced by Insulin, dexamethasone and IBMX, and these were used as in vitro models of adipogenesis. The effects of cordycepin on adipogenesis were examined with particular focus on the regulation of CCAAT/enhancer-binding protein β (C/EBPβ) and PPARγ. KEY RESULTS Cordycepin suppressed the lipid accumulation and induction of adipogenic markers that occurred on differentiation of pre-adipocytes and also blocked the down-regulation of a pre-adipocyte marker. This anti-adipogenic effect was reversible and mediated by an adenosine transporter, but not A(1) , A(2) or A(3) adenosine receptors. This effect of cordycepin was not reproduced by other adenosine-related substances, including ATP, ADP and adenosine. Early induction of the adipogenic C/EBPβ-PPARγ pathway was suppressed by cordycepin. Blockade of mTORC1 via inhibition of PKB (Akt) and activation of AMP kinase was identified as the crucial upstream event targeted by cordycepin. In addition to its negative effect on adipogenesis, cordycepin suppressed lipid accumulation in mature adipocytes. CONCLUSIONS AND IMPLICATIONS These results suggest that the anti-adipogenic effects of cordycepin occur through its intervention in the mTORC1-C/EBPβ-PPARγ pathway. Cordycepin, by blocking both adipogenesis and lipid accumulation, may have potential as a therapeutic agent for effective treatment of obesity and obesity-related disorders.
    British Journal of Pharmacology 04/2012; 167(3):561-75. DOI:10.1111/j.1476-5381.2012.02005.x · 4.84 Impact Factor
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    H Kato · S Nakajima · Y Saito · S Takahashi · R Katoh · M Kitamura ·
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    ABSTRACT: Mammalian target of rapamycin (mTOR) has a key role in the regulation of an array of cellular function. We found that rapamycin, an inhibitor of mTOR complex 1 (mTORC1), attenuated endoplasmic reticulum (ER) stress-induced apoptosis. Among three major branches of the unfolded protein response, rapamycin selectively suppressed the IRE1–JNK signaling without affecting PERK and ATF6 pathways. ER stress rapidly induced activation of mTORC1, which was responsible for induction of the IRE1–JNK pathway and apoptosis. Activation of mTORC1 reduced Akt phosphorylation, which was an event upstream of IRE–JNK signaling and consequent apoptosis. In vivo, administration with rapamycin significantly suppressed renal tubular injury and apoptosis in tunicamycin-treated mice. It was associated with enhanced phosphorylation of Akt and suppression of JNK activity in the kidney. These results disclosed that, under ER stress conditions, mTORC1 causes apoptosis through suppression of Akt and consequent induction of the IRE1–JNK pathway.
    Cell death and differentiation 02/2012; 19(2):310-320. DOI:10.1038/cdd.2011.98 · 8.18 Impact Factor
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    ABSTRACT: Subtilase cytotoxin (SubAB) is the prototype of a distinct AB(5) toxin family produced by Shiga toxigenic Escherichia coli. Recent reports disclosed pro-apoptotic pathways triggered by SubAB, whereas its anti-apoptotic signals have not been elucidated. In the present study, we investigated pro-survival signaling elicited by SubAB, especially focusing on extracellular signal-regulated kinase (ERK) and Akt. We found that SubAB activated ERK and Akt, and inhibition of individual kinases enhanced SubAB-triggered apoptosis. SubAB induced endoplasmic reticulum (ER) stress, and other ER stress inducers mimicked the stimulatory effects of SubAB on ERK and Akt. Attenuation of ER stress reduced SubAB-induced phosphorylation of these kinases, suggesting involvement of the unfolded protein response (UPR). SubAB induced activation of protein kinase-like ER kinase (PERK) and phosphorylation of eukaryotic translation initiation factor 2α (eIF2α), and phosphorylation of eIF2α by salubrinal caused activation of ERK and Akt, leading to cell survival. Dominant-negative inhibition of PERK enhanced SubAB-induced apoptosis and reduced phosphorylation of ERK and Akt. Furthermore, the anti-apoptotic effect of eIF2α was significantly reversed by inhibition of ERK and Akt. These results suggest cytoprotective roles of ERK and Akt in SubAB-triggered, ER stress-mediated apoptosis.
    Biochemical and Biophysical Research Communications 06/2011; 410(4):852-8. DOI:10.1016/j.bbrc.2011.06.078 · 2.30 Impact Factor
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    ABSTRACT: Cigarette smoke triggers apoptosis through oxidative stress- and endoplasmic reticulum (ER) stress-dependent induction of CCAAT/enhancer-binding protein-homologous protein (CHOP) (Tagawa et al., 2008. Free Radic. Biol. Med. 45, 50-59). We investigated roles of individual reactive oxygen/nitrogen species in the transcriptional induction of CHOP by cigarette smoke. Exposure of bronchial epithelial cells to O(2)(-), ONOO(-) or H(2)O(2) induced expression of CHOP, whereas NO alone did not. Induction of CHOP mRNA by cigarette smoke extract (CSE) was attenuated by scavengers for O(2)(-), ONOO(-) or NO, whereas scavenging H(2)O(2) did not affect the induction of CHOP. Like CSE, O(2)(-) and ONOO(-) caused activation of the CHOP gene promoter. Scavengers for O(2)(-), ONOO(-) or NO attenuated CSE-triggered activation of the CHOP gene promoter. CSE, O(2)(-) and ONOO(-) induced phosphorylation of protein kinase-like ER kinase (PERK) and eukaryotic translation initiation factor 2α (eIF2α) and caused induction of downstream activating transcription factor 4 (ATF4). Scavengers for O(2)(-), ONOO(-) or NO attenuated induction of ATF4 by CSE. Furthermore, dominant-negative inhibition of the PERK-eIF2α pathway exclusively suppressed CSE-triggered induction of CHOP and consequent apoptosis. These results suggest that O(2)(-) and ONOO(-) are selectively involved in CSE-triggered induction of CHOP and that the PERK-eIF2α pathway plays a crucial role in the induction of CHOP and apoptosis downstream of the particular reactive oxygen species.
    Toxicology 06/2011; 287(1-3):105-12. DOI:10.1016/j.tox.2011.06.005 · 3.62 Impact Factor
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    ABSTRACT: Proteasome inhibitor MG132 blocks activation of NF-κB by preventing degradation of IκB. In this report, we propose an alternative mechanism by which MG132 inhibits cytokine-triggered NF-κB activation. We found that MG132 induced endoplasmic reticulum (ER) stress, and attenuation of ER stress blunted the suppressive effect of MG132 on NF-κB. Through ER stress, MG132 up-regulated C/EBPβ mRNA transiently and caused sustained accumulation of its translational products liver activating protein (LAP) and liver-enriched inhibitory protein (LIP), both of which were identified as suppressors of NF-κB. Our results disclosed a novel mechanism underlying inhibition of NF-κB by MG132.
    FEBS letters 05/2011; 585(14):2249-54. DOI:10.1016/j.febslet.2011.05.047 · 3.17 Impact Factor
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    M Kitamura · H Kato · Y Saito · S Nakajima · S Takahashi · H Johno · L Gu · R Katoh ·
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    ABSTRACT: The unfolded protein response (UPR) is involved in a diverse range of pathologies triggered by endoplasmic reticulum (ER) stress. Endeavor to seek selective regulators of the UPR is a promising challenge towards therapeutic intervention in ER stress-related disorders. In the present report, we describe aberrant, differential and bidirectional regulation of the UPR by 3'-deoxyadenosine (cordycepin) towards cell survival. 3'-Deoxyadenosine blocked ER stress-induced apoptosis via inhibiting the IRE1-JNK pro-apoptotic pathway. 3'-Deoxyadenosine also inhibited apoptosis through reinforcement of the pro-survival eIF2α signaling without affecting PERK activity. It was associated with depression of GADD34 that dephosphorylates eIF2α, and dephosphorylation of eIF2α by salubrinal mimicked the anti-apoptotic effect of 3'-deoxyadenosine. Unexpectedly, although 3'-deoxyadenosine caused activation of eIF2α, it inhibited downstream pro-apoptotic events including induction of ATF4 and expression of CHOP. Cooperation of adenosine transporter and A3 adenosine receptor, but not A1/A2 receptors, mediated the pluripotent effects of 3'-deoxyadenosine. In mice, ER stress caused activation of JNK, expression of CHOP and induction of apoptosis in renal tubules. The apoptosis was significantly attenuated by administration with 3'-deoxyadenosine, and it was correlated with blunted induction of JNK and CHOP in the kidney. These results disclosed atypical pro-survival regulation of the UPR by 3'-deoxyadenosine, which may be advantageous for the treatment of intractable, ER stress-related disorders.
    Cell death and differentiation 05/2011; 18(12):1876-88. DOI:10.1038/cdd.2011.63 · 8.18 Impact Factor
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    ABSTRACT: Subtilase cytotoxin (SubAB) that selectively cleaves BiP/GRP78 triggers the unfolded protein response (UPR) and protects mice from endotoxic lethality and collagen arthritis. We found that pretreatment of cells with SubAB suppressed tumor necrosis alpha (TNF-α)-induced activation of NF-κB and NF-κB-dependent chemokine expression. To elucidate underlying mechanisms, the involvement of C/EBP and Akt, putative regulators of NF-κB, was investigated. Among members of the C/EBP family, SubAB preferentially induced C/EBPβ. Overexpression of C/EBPβ suppressed TNF-α-induced NF-κB activation, and knockdown of C/EBPβ attenuated the suppressive effect of SubAB on NF-κB. We identified that the ATF6 branch of the UPR plays a crucial role in the induction of C/EBPβ. In addition to this effect, SubAB depressed basal and TNF-α-induced phosphorylation of Akt via the UPR. It was mediated by the induction of ATF6 and consequent activation of mTOR that dephosphorylated Akt. Inhibition of Akt attenuated activation of NF-κB by TNF-α, suggesting that the mTOR-Akt pathway is another target for SubAB-initiated, UPR-mediated NF-κB suppression. These results elucidated that SubAB blunts activation of NF-κB through ATF6-dependent mechanisms, i.e., preferential induction of C/EBPβ and mTOR-dependent dephosphorylation of Akt.
    Molecular and Cellular Biology 02/2011; 31(8):1710-8. DOI:10.1128/MCB.00939-10 · 4.78 Impact Factor
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    ABSTRACT: Recent reports suggested involvement of mitogen-activated protein (MAP) kinases in the pathogenesis of Shiga toxin-induced hemolytic uremic syndrome (HUS). In the present study, we investigated a role for subtilase cytotoxin (SubAB), a possible trigger for HUS, in the regulation of MAP kinases. Treatment of cells with SubAB caused phosphorylation of c-Jun NH(2)-terminal kinase, extracellular signal-regulated kinase (ERK), and p38 MAP kinase. It was associated with activation of activator protein 1 (AP-1) and induction of AP-1-dependent transcription. SubAB induced the unfolded protein response (UPR) and consequently caused MAP kinase activation. SubAB led to induction of three major branches of the UPR, and the protein kinase-like endoplasmic reticulum kinase and inositol-requiring ER-to-nucleus signal kinase 1 pathways were responsible for the activation of MAP kinases. These results elucidated the potential of SubAB to trigger MAP kinase pathways via the UPR, which may contribute to the pathogenesis of Shiga toxin-induced HUS.
    Toxicological Sciences 12/2010; 120(1):79-86. DOI:10.1093/toxsci/kfq368 · 3.85 Impact Factor
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    ABSTRACT: Tumor necrosis factor-α (TNF-α) is a key regulator of adipose tissue mass, but mechanisms underlying this effect have not been fully elucidated. We found that exposure to TNF-α caused a significant decrease in the number of adipocytes, but not preadipocytes. Subsequent experiments revealed that TNF-α selectively deleted adipocytes through induction of apoptosis. Following exposure to TNF-α, rapid activation of nuclear factor-B (NF-B) was observed only in preadipocytes, but not in adipocytes. Inhibition of NF-B rendered preadipocytes susceptible to TNF-α-induced apoptosis, suggesting that different activity of NF-B is the determinant for the distinct apoptotic responses. During adipocyte differentiation, expression and activity of peroxisome proliferator-activated receptor-γ (PPARγ) were upregulated. Treatment of preadipocytes with a PPARγ agonist attenuated NF-B activation and rendered the cells vulnerable to TNF-α-induced apoptosis. Conversely, treatment of adipocytes with a PPARγ antagonist enhanced NF-B activation and conferred resistance to TNF-α-induced apoptosis, suggesting involvement of PPARγ in the suppression of NF-B in adipocytes. We also found that, following differentiation, expression and activity of CCAAT/enhancer binding protein (C/EBP), especially C/EBPα and C/EBPΒ, were upregulated in adipocytes. Overexpression of individual C/EBPs significantly inhibited activation of NF-B in preadipocytes. Furthermore, transfection with siRNA for C/EBPα or C/EBPΒ enhanced activity of NF-B in adipocytes, suggesting that C/EBP is also involved in the repression of NF-B in adipocytes. These results suggested novel mechanisms by which TNF-α selectively deletes adipocytes in the adipose tissue. The C/EBP- and PPARγ-mediated suppression of NF-B may contribute to TNF-α-related loss of adipose tissue mass under certain pathological situations, such as cachexia.
    Laboratory Investigation 09/2010; 90(9):1385-95. DOI:10.1038/labinvest.2010.118 · 3.68 Impact Factor
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    ABSTRACT: We recently reported that subtilase cytotoxin (SubAB) has the potential to attenuate experimental models of inflammatory diseases [3]. Currently, little is known about underlying mechanisms involved in this therapeutic effect. In the present report, we show that SubAB induces A20, the endogenous negative regulator of NF-kappaB, in vitro and in vivo. This stimulatory effect occurred at the transcriptional level, and SubAB induced activation of the A20 promoter. We found that, in the early phase, SubAB triggered activation of NF-kappaB in a dose-dependent manner. Blockade of NF-kappaB abrogated expression of A20 by SubAB. SubAB rapidly triggered the unfolded protein response (UPR), and induction of the UPR by other agents (thapsigargin and A23187) mimicked the stimulatory effects of SubAB, both on NF-kappaB and on A20. The induction of A20 by thapsigargin was correlated with activation of the A20 promoter, which was not observed in the kappaB-mutated A20 promoter. Furthermore, induction of A20 by SubAB was substantially attenuated by treatment with different chemical chaperones. These results elucidated for the first time that the anti-inflammatory SubAB has the potential to induce A20 through the UPR-NF-kappaB-dependent pathway.
    Biochemical and Biophysical Research Communications 06/2010; 397(2):176-80. DOI:10.1016/j.bbrc.2010.05.069 · 2.30 Impact Factor

Publication Stats

419 Citations
110.66 Total Impact Points


  • 2009-2015
    • University of Yamanashi
      • Interdisciplinary Graduate School of Medicine and Engineering
      Kōhu, Yamanashi, Japan
  • 2011
    • University of Adelaide
      • Research Centre for Infectious Diseases (RCID)
      Tarndarnya, South Australia, Australia