Publications by authors named "Ken Itoh"

139 Publications

Calpain-1 C2L domain peptide protects mouse hippocampus-derived neuronal HT22 cells against glutamate-induced oxytosis.

Biochem Biophys Rep 2021 Sep 12;27:101101. Epub 2021 Aug 12.

Laboratory of Cell Biochemistry, Department of Biological Science, Graduate School of Science and Engineering, Iwate University, 4-3-5 Ueda, Morioka, Iwate, 020-8551, Japan.

Calpains are Ca-dependent cysteine proteases; their aberrant activation is associated with several neurodegenerative diseases. The μ-calpain catalytic subunit, calpain-1, is located in the cytoplasm as well as in the mitochondria. Mitochondrial calpain-1 cleaves apoptosis-inducing factor (AIF), leading to apoptotic cell death. We have previously reported that short peptides of calpain-1 C2-like domain conjugated with cell penetrating peptide HIV-Tat (Tat-μCL) selectively inhibit mitochondrial calpain-1 and effectively prevent neurodegenerative diseases of the eye. In this study, we determined whether mitochondrial calpain-1 mediates oxytosis (oxidative glutamate toxicity) in hippocampal HT22 cells using Tat-μCL and newly generated polyhistidine-conjugated μCL peptide and compared their efficacies in preventing oxytosis. TUNEL assay and single strand DNA staining revealed that both μCL peptides inhibited glutamate-induced oxytosis. Additionally, both the peptides suppressed the mitochondrial AIF translocation into the nucleus. All polyhistidine-μCL peptides (containing 4-16 histidine residues) showed higher cell permeability than Tat-μCL. Notably, tetrahistidine (H4)-μCL exerted the highest cytoprotective activity. Thus, H4-μCL may be a potential peptide drug for calpain-1-mediated neurodegenerative diseases such as Alzheimer's disease.
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http://dx.doi.org/10.1016/j.bbrep.2021.101101DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8374356PMC
September 2021

Distinct Regulations of Gene Expression for Stress Response and Substrate Induction.

Mol Cell Biol 2021 Aug 16:MCB0023621. Epub 2021 Aug 16.

Departments of Medical Biochemistry, Tohoku University Graduate School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai 980-8575, Japan.

Heme oxygenase-1 (HO-1) is the key enzyme for heme catabolism and cytoprotection. Whereas gene expression in response to various stresses has been investigated extensively, the precise mechanisms by which gene expression is regulated by the HO-1 substrate heme remain elusive. To systematically examine whether stress-mediated induction and substrate-mediated induction of utilize similar or distinct regulatory pathways, we developed an HO-1-DsRed-knock-in reporter mouse in which the gene is floxed by loxP sites and the gene has been inserted. Myeloid lineage-specific recombination of the floxed locus led to fluorescence derived from expression of the HO-1-DsRed fusion protein in peritoneal macrophages. We also challenged general recombination of the locus and generated mice harboring heterozygous recombinant alleles, which enabled us to monitor HO-1-DsRed expression in the whole body and . HO-1 inducers upregulated HO-1-DsRed expression in myeloid lineage cells isolated from the mice. Notably, analyses of peritoneal macrophages from HO-1-DsRed mice lacking NRF2, a major regulator of the oxidative/electrophilic stress response, led us to identify NRF2-dependent stress response-mediated induction and NRF2-independent substrate-mediated induction. Thus, the gene is subjected to at least two distinct levels of regulation, and the available lines of evidence suggest that substrate induction in peritoneal macrophages is independent of CNC family-based regulation.
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http://dx.doi.org/10.1128/MCB.00236-21DOI Listing
August 2021

Capillary Electrophoresis Mass Spectrometry-Based Metabolomics of Plasma Samples from Healthy Subjects in a Cross-Sectional Japanese Population Study.

Metabolites 2021 May 13;11(5). Epub 2021 May 13.

Department of Metabolomics Innovation, Hirosaki University Graduate School of Medicine, 5 Zaifu-cho, Hirosaki 036-8562, Japan.

For large-scale metabolomics, such as in cohort studies, normalization protocols using quality control (QC) samples have been established when using data from gas chromatography and liquid chromatography coupled to mass spectrometry. However, normalization protocols have not been established for capillary electrophoresis-mass spectrometry metabolomics. In this study, we performed metabolome analysis of 314 human plasma samples using capillary electrophoresis-mass spectrometry. QC samples were analyzed every 10 samples. The results of principal component analysis for the metabolome data from only the QC samples showed variations caused by capillary replacement in the first principal component score and linear variation with continuous measurement in the second principal component score. Correlation analysis between diagnostic blood tests and plasma metabolites normalized by the QC samples was performed for samples from 188 healthy subjects who participated in a Japanese population study. Five highly correlated pairs were identified, including two previously unidentified pairs in normal healthy subjects of blood urea nitrogen and guanidinosuccinic acid, and gamma-glutamyl transferase and cysteine glutathione disulfide. These results confirmed the validity of normalization protocols in capillary electrophoresis-mass spectrometry using large-scale metabolomics and comprehensive analysis.
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http://dx.doi.org/10.3390/metabo11050314DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8153282PMC
May 2021

Health improvement framework for actionable treatment planning using a surrogate Bayesian model.

Nat Commun 2021 05 25;12(1):3088. Epub 2021 May 25.

Department of Biomedical Data Intelligence, Graduate School of Medicine, Kyoto University, Kyoto, Japan.

Clinical decision-making regarding treatments based on personal characteristics leads to effective health improvements. Machine learning (ML) has been the primary concern of diagnosis support according to comprehensive patient information. A prominent issue is the development of objective treatment processes in clinical situations. This study proposes a framework to plan treatment processes in a data-driven manner. A key point of the framework is the evaluation of the actionability for personal health improvements by using a surrogate Bayesian model in addition to a high-performance nonlinear ML model. We first evaluate the framework from the viewpoint of its methodology using a synthetic dataset. Subsequently, the framework is applied to an actual health checkup dataset comprising data from 3132 participants, to lower systolic blood pressure and risk of chronic kidney disease at the individual level. We confirm that the computed treatment processes are actionable and consistent with clinical knowledge for improving these values. We also show that the improvement processes presented by the framework can be clinically informative. These results demonstrate that our framework can contribute toward decision-making in the medical field, providing clinicians with deeper insights.
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http://dx.doi.org/10.1038/s41467-021-23319-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8149666PMC
May 2021

Genetic ablation of Nrf2 exacerbates neurotoxic effects of acrylamide in mice.

Toxicology 2021 05 16;456:152785. Epub 2021 Apr 16.

Department of Occupational and Environmental Health, Tokyo University of Science, Noda, Japan. Electronic address:

Acrylamide (ACR), a recognized neurotoxicant in humans and experimental animals, is widely used in industry and in food generated through Maillard reaction. Nuclear factor erythroid 2-related factor 2 (Nrf2) is a master regulator of the cellular defense system and activates antioxidants and cytoprotective genes. The exact roles of Nrf2 in environmental electrophile-induced neurotoxicity is poorly understood. The aim of this study was to determine the roles of Nrf2 in ACR-induced neurotoxicity including degeneration of monoaminergic axons and sensorimotor dysfunction. Male 10-week-old C57BL/6JJcl Nrf2-knockout mice and wild type (WT) counterparts were each divided into four groups of 12 and provided with drinking water containing acrylamide at 0, 67, 110 or 200 ppm for four weeks. The effects of acrylamide were examined by landing foot spread test, immunohistochemistry for noradrenaline (NA) and serotonin (5-HT)-containing axons and Iba1-positive microglia in the prefrontal cortex as well as quantitative real-time polymerase chain reaction (qRT-PCR) on antioxidant, proinflammatory and anti-inflammatory genes in the prefrontal cortex. Relative to the wild type, exposure of Nrf2-knockout mice to acrylamide increased hindlimb splay length, microglial area and process length as well as decreasing the density of NA and 5-HT-immunoreactive axons to a greater extent. Moreover, deletion of Nrf2 gene suppressed acrylamide-induced mRNA upregulation of Nrf2-antioxidants, NAD(P): quinone oxidoreductase 1 (NQO1), superoxide dismutase-1 (SOD-1) and heme oxygenase-1 (HO-1) as well as anti-inflammatory markers such as, arginase-1 (Arg1), found in the inflammatory zone-1 (Fizz1), chitinase-like 3 (Chi3l3), interleukin-4 receptor alpha (IL-4Rα), cluster of differentiation  206 (CD206) and transforming growth factor beta-1 (TGFβ1) while enhancing acrylamide-induced upregulation of pro-inflammatory cytokines, interleukin-1 beta (IL-1β), tumor necrosis-alpha (TNF-α) and inducible nitric oxide synthase (iNOS) in the prefrontal cortex. The results demonstrate susceptibility of mice lacking the Nrf2 gene to acrylamide-induced neurotoxicity and neuroinflammation with the activation of microglia. Moreover, the results suggest the role of Nrf2 not only in induction of antioxidant gene expression, but also in suppression of proinflammatory cytokine gene expression.
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http://dx.doi.org/10.1016/j.tox.2021.152785DOI Listing
May 2021

The CD36 Ligand-Promoted Autophagy Protects Retinal Pigment Epithelial Cells from Oxidative Stress.

Oxid Med Cell Longev 2021 28;2021:6691402. Epub 2021 Mar 28.

Faculty of Pharmacy, Université de Montréal, 2940 Chemin de Polytechnique, Montreal, QC, Canada H3T 1J4.

The retinal pigment epithelium (RPE) performs many functions that maintain photoreceptor health. Oxidative damage to the RPE is a critical component in the pathogenesis of eye diseases such as age-related macular degeneration (AMD). Ligands of the cluster of differentiation 36 (CD36) have previously preserved photoreceptor integrity in mouse models of AMD. The cytoprotective effect of the CD36 ligand MPE-001 on RPE cells has now been elucidated employing a model of oxidative stress. Sodium iodate (NaIO) induced formation of reactive oxygen species and apoptosis in human RPE cells, which were decreased by MPE-001 without affecting antioxidant enzyme transcription. Immunoblotting and immunostaining assays showed a restorative effect of MPE-001 on the autophagic flux disrupted by NaIO, which was associated with an increase in syntaxin 17-positive mature autophagosomes. The cytoprotective effect of MPE-001 was completely abolished by the autophagy inhibitors wortmannin and bafilomycin A1. In conclusion, we report for the first time an autophagy-dependent protection of RPE cells from oxidative stress by a CD36 ligand.
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http://dx.doi.org/10.1155/2021/6691402DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8019622PMC
June 2021

Association between Serum Concentration of Carotenoid and Visceral Fat.

Nutrients 2021 Mar 11;13(3). Epub 2021 Mar 11.

Innovation Center for Health Promotion, Graduate School of Medicine, Hirosaki University, 5 Zaifu-cho, Hirosaki, Aomori 036-8562, Japan.

Consumption of fruits and vegetables rich in carotenoids has been widely reported to prevent cardiovascular diseases. However, the relationship between serum carotenoid concentrations and visceral fat area (VFA), which is considered a better predictor of cardiovascular diseases than the body-mass index (BMI) and waist circumference, remains unclear. Therefore, we examined the relationship in healthy individuals in their 20s or older, stratified by sex and age, to compare the relationship between serum carotenoid concentrations and VFA and BMI. The study was conducted on 805 people, the residents in Hirosaki city, Aomori prefecture, who underwent a health checkup. An inverse relationship between serum carotenoid concentrations and VFA and BMI was observed only in women. In addition, the results were independent of the intake of dietary fiber, which is mainly supplied from vegetables as well as carotenoids. This suggests that consumption of a diet rich in carotenoids (especially lutein and beta-carotene) is associated with lower VFA, which is a good predictor of cardiovascular disease, especially in women. This study is the first to comprehensively evaluate the association between serum carotenoid levels and VFA in healthy individuals.
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http://dx.doi.org/10.3390/nu13030912DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7999533PMC
March 2021

Characterization of mitochondrial calpain-5.

Biochim Biophys Acta Mol Cell Res 2021 04 16;1868(5):118989. Epub 2021 Feb 16.

Laboratory of Cell Biochemistry, Department of Biological Science, Graduate School of Science and Engineering, Iwate University, 4-3-5 Ueda, Morioka, Iwate 020-8551, Japan. Electronic address:

Calpain, a Ca-dependent cysteine protease, plays a significant role in gene expression, signal transduction, and apoptosis. Mutations in human calpain-5 cause autosomal dominant neovascular inflammatory vitreoretinopathy and the inhibition of calpain-5 activity may constitute an effective therapeutic strategy for this condition. Although calpain-5 is ubiquitously expressed in mammalian tissues and was recently found to be present in the mitochondria as well as in the cytosol, its physiological function and enzymological properties require further elucidation. The objective of the current study was to determine the characteristics of mitochondrial calpain-5 in porcine retinas, human HeLa cells, and C57BL/6J mice using subcellular fractionation. We found that mitochondrial calpain-5 was proteolyzed/autolyzed at low Ca concentrations in mitochondria isolated from porcine retinas and by thapsigargin-induced endoplasmic reticulum (ER) stress in HeLa cells. Further, mitochondrial calpain-5, as opposed to cytosolic calpain-5, was activated during the early stages of ER stress in C57BL/6J mice. These results showed that mitochondrial calpain-5 was activated at low Ca concentrations in vitro and in response to ER stress in vivo. The present study provides new insights into a novel calpain system in the mitochondria that includes stress responses during the early phases of ER stress. Further, activation of mitochondrial calpain-5 by treatment using low-molecular-weight compounds may have therapeutic potential for diseases related to ER stress, including neurodegenerative diseases, metabolic syndromes, diabetes, and cancer.
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http://dx.doi.org/10.1016/j.bbamcr.2021.118989DOI Listing
April 2021

Correction to: Prevalence of the mitochondrial 1555 A>G and 1494 C>T mutations in a community-dwelling population in Japan.

Hum Genome Var 2020 27;7:36. Epub 2020 Oct 27.

Department of Otorhinolaryngology, Hirosaki University Graduate School of Medicine, Hirosaki, Japan.

[This corrects the article DOI: 10.1038/s41439-020-00115-9.].
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http://dx.doi.org/10.1038/s41439-020-00123-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7591919PMC
October 2020

JDP2 is directly regulated by ATF4 and modulates TRAIL sensitivity by suppressing the ATF4-DR5 axis.

FEBS Open Bio 2020 12 13;10(12):2771-2779. Epub 2020 Nov 13.

Department of Stress Response Science, Center for Advanced Medical Research, Hirosaki University Graduate School of Medicine, Japan.

Jun dimerization protein 2 (JDP2) is a bZip-type transcription factor, which acts as a repressor or activator of several cellular processes, including cell differentiation and chromatin remodeling. Previously, we found that a stress-responsive transcription factor, known as activating transcription factor 4 (ATF4), enhances JDP2 gene expression in human astrocytoma U373MG and cervical cancer HeLa cells; however, the role of JDP2 in the ATF4-mediated stress response remained unclear. Here, we reported that siRNA-mediated JDP2 knockdown enhances the expression of several ATF4 target genes, including ASNS, and death receptors 4 and 5 (DR4 and DR5) in HeLa cells. In addition, the results of a transient reporter assay indicate that JDP2 overexpression represses ER stress-mediated DR5 promoter activation suggesting that JDP2 negatively regulates ATF4-mediated gene expression. Curiously, knockdown of JDP2 increases the sensitivity of cells to TNF-related apoptosis-inducing ligand (TRAIL), which induces apoptosis in cancer cells through DR4 and DR5. These results indicate that JDP2 functions as a negative feedback regulator of the ATF4 pathway and contributes to TRAIL resistance in cancer cells.
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http://dx.doi.org/10.1002/2211-5463.13017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7714084PMC
December 2020

Prevalence of the mitochondrial 1555 A>G and 1494 C>T mutations in a community-dwelling population in Japan.

Hum Genome Var 2020 18;7:27. Epub 2020 Sep 18.

Department of Otorhinolaryngology, Hirosaki University Graduate School of Medicine, Hirosaki, Japan.

Single nucleotide polymorphisms in mitochondrial DNA, such as mitochondrial 1555 A>G (m.1555 A>G) and mitochondrial 1494 C>T (m.1494 C>T), are known to be causative mutations of nonsyndromic hearing loss following exposure to aminoglycoside antibiotics. The prevalence of the m.1555 A>G and m.1494 C>T mutations has not been reported for the general population in Japan. The purpose of this study was to investigate the prevalence of m.1555 A>G and m.1494 C>T mutations in a community-dwelling population in Japan in order to prevent aminoglycoside-induced hearing loss. We recruited participants older than 20 years of age to the Iwaki Health Promotion Project in 2014, 2015, and 2016, resulting in the recruitment of 1,683 participants. For each participant, we performed a hearing test and a genetic test for the m.1555 A>G and m.1494 C>T mutations using the TaqMan genotyping method. The m.1555 A>G mutation was detected in only 1 of the 1,683 participants (0.06%). This carrier of the m.1555 A>G mutation was a 69-year-old male with bilateral, symmetric, and high-frequency hearing loss. We provided genetic counseling and distributed a drug card advising him to avoid the administration of aminoglycoside antibiotics. In contrast, the m.1494 C>T mutation was not detected in this study population.
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http://dx.doi.org/10.1038/s41439-020-00115-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7501278PMC
September 2020

Association of single nucleotide polymorphisms in the NRF2 promoter with vascular stiffness with aging.

PLoS One 2020 11;15(8):e0236834. Epub 2020 Aug 11.

Department of Stress Response Science, Center for Advanced Medical Research, Hirosaki University Graduate School of Medicine, Hirosaki, Japan.

Purpose: Pulse wave velocity (PWV), an indicator of vascular stiffness, increases with age and is increasingly recognized as an independent risk factor for cardiovascular disease (CVD). Although many mechanical and chemical factors underlie the stiffness of the elastic artery, genetic risk factors related to age-dependent increases in PWV in apparently healthy people are largely unknown. The transcription factor nuclear factor E2 (NF-E2)-related factor 2 (Nrf2), which is activated by unidirectional vascular pulsatile shear stress or oxidative stress, regulates vascular redox homeostasis. Previous reports have shown that a SNP in the NRF2 gene regulatory region (-617C>A; hereafter called SNP-617) affects NRF2 gene expression such that the minor A allele confers lower gene expression compared to the C allele, and it is associated with various diseases, including CVD. We aimed to investigate whether SNP-617 affects vascular stiffness with aging in apparently healthy people.

Methods: Analyzing wide-ranging data obtained from a public health survey performed in Japan, we evaluated whether SNP-617 affected brachial-ankle PWV (baPWV) in never-smoking healthy subjects (n = 642). We also evaluated the effects of SNP-617 on other cardiovascular and blood test measurements.

Results: We have shown that not only AA carriers (n = 55) but also CA carriers (n = 247) show arterial stiffness compared to CC carriers (n = 340). Furthermore, SNP-617 also affected blood pressure indexes such as systolic blood pressure and mean arterial pressure but not the ankle brachial pressure index, an indicator of atherosclerosis. Multivariate analysis showed that SNP-617 accelerates the incremental ratio of baPWV with age.

Conclusions: This study is the first to show that SNP-617 affects the age-dependent increase in vascular stiffness. Our results indicate that low NRF2 activity induces premature vascular aging and could be targeted for the prevention of cardiovascular diseases associated with aging.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0236834PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7418968PMC
October 2020

Association between Biomarkers of Cardiovascular Diseases and the Blood Concentration of Carotenoids among the General Population without Apparent Illness.

Nutrients 2020 Jul 31;12(8). Epub 2020 Jul 31.

Graduate School of Medicine, Hirosaki University, 5 Zaifu-cho, Hirosaki, Aomori 036-8562, Japan.

Several studies have demonstrated that carotenoid-rich vegetables are useful against cardiovascular diseases (CVDs). However, it is still unclear when a healthy population should start eating these vegetables to prevent CVDs. In this study, we evaluated the role of carotenoids in CVD markers in healthy subjects using age-stratified analysis. We selected 1350 subjects with no history of apparent illness who were undergoing health examinations. We then evaluated the relationship between the serum concentrations of six major carotenoids as well as their total, and nine CVD markers (i.e., body mass index (BMI), pulse wave velocity (PWV), systolic blood pressure (SBP), diastolic blood pressure (DBP), Homeostatic Model Assessment of Insulin Resistance (HOMA-IR), blood insulin, fasting blood glucose (FBG), triglycerides (TGs), and high-density lipoprotein (HDL) cholesterol) using multiple regression analysis. It was found that the total carotenoid level was significantly associated with seven markers other than BMI and FBG in males and with eight markers other than DBP in females. Many of these relationships were independent of lifestyle habits. Many significant relationships were found in young males (aged 20-39) and middle-aged females (aged 40-59). These findings can be used as lifestyle guidance for disease prevention although the causal relationships should be confirmed.
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http://dx.doi.org/10.3390/nu12082310DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7469056PMC
July 2020

Blockade of PAR-1 Signaling Attenuates Cardiac Hypertrophy and Fibrosis in Renin-Overexpressing Hypertensive Mice.

J Am Heart Assoc 2020 06 4;9(12):e015616. Epub 2020 Jun 4.

Department of Cardiology and Nephrology Hirosaki University Graduate School of Medicine Hirosaki Japan.

Background Although PAR-1 (protease-activated receptor-1) exerts important functions in the pathophysiology of the cardiovascular system, the role of PAR-1 signaling in heart failure development remains largely unknown. We tested the hypothesis that PAR-1 signaling inhibition has protective effects on the progression of cardiac remodeling induced by chronic renin-angiotensin system activation using renin-overexpressing hypertensive (Ren-Tg) mice. Methods and Results We treated 12- to 16-week-old male wild-type (WT) mice and Ren-Tg mice with continuous subcutaneous infusion of the PAR-1 antagonist SCH79797 or vehicle for 4 weeks. The thicknesses of interventricular septum and the left ventricular posterior wall were greater in Ren-Tg mice than in WT mice, and SCH79797 treatment significantly decreased these thicknesses in Ren-Tg mice. The cardiac fibrosis area and monocyte/macrophage deposition were greater in Ren-Tg mice than in WT mice, and both conditions were attenuated by SCH79797 treatment. Cardiac mRNA expression levels of PAR-1, TNF-α (tumor necrosis factor-α), TGF-β1 (transforming growth factor-β1), and COL3A1 (collagen type 3 α1 chain) and the ratio of β-myosin heavy chain (β-MHC) to α-MHC were all greater in Ren-Tg mice than in WT mice; SCH79797 treatment attenuated these increases in Ren-Tg mice. Prothrombin fragment 1+2 concentration and factor Xa in plasma were greater in Ren-Tg mice than in WT mice, and both conditions were unaffected by SCH79797 treatment. In isolated cardiac fibroblasts, both thrombin and factor Xa enhanced ERK1/2 (extracellular signal-regulated kinase 1/2) phosphorylation, and SCH79797 pretreatment abolished this enhancement. Furthermore, gene expression of PAR-1, TGF-β1, and COL3A1 were enhanced by factor Xa, and all were inhibited by SCH79797. Conclusions The results indicate that PAR-1 signaling is involved in cardiac remodeling induced by renin-angiotensin system activation, which may provide a novel therapeutic target for heart failure.
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http://dx.doi.org/10.1161/JAHA.119.015616DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7429042PMC
June 2020

Ribosome binding protein GCN1 regulates the cell cycle and cell proliferation and is essential for the embryonic development of mice.

PLoS Genet 2020 04 23;16(4):e1008693. Epub 2020 Apr 23.

Department of Stress Response Science, Center for Advanced Medical Research, Hirosaki University, Hirosaki, Japan.

Amino acids exert many biological functions, serving as allosteric regulators and neurotransmitters, as constituents in proteins and as nutrients. GCN2-mediated phosphorylation of eukaryotic initiation factor 2 alpha (elF2α) restores homeostasis in response to amino acid starvation (AAS) through the inhibition of the general translation and upregulation of amino acid biosynthetic enzymes and transporters by activating the translation of Gcn4 and ATF4 in yeast and mammals, respectively. GCN1 is a GCN2-binding protein that possesses an RWD binding domain (RWDBD) in its C-terminus. In yeast, Gcn1 is essential for Gcn2 activation by AAS; however, the roles of GCN1 in mammals need to be established. Here, we revealed a novel role of GCN1 that does not depend on AAS by generating two Gcn1 mutant mouse lines: Gcn1-knockout mice (Gcn1 KO mice (Gcn1-/-)) and RWDBD-deleted mutant mice (Gcn1ΔRWDBD mice). Both mutant mice showed growth retardation, which was not observed in the Gcn2 KO mice, such that the Gcn1 KO mice died at the intermediate stage of embryonic development because of severe growth retardation, while the Gcn1ΔRWDBD embryos showed mild growth retardation and died soon after birth, most likely due to respiratory failure. Extension of pregnancy by 24 h through the administration of progesterone to the pregnant mothers rescued the expression of differentiation markers in the lungs and prevented lethality of the Gcn1ΔRWDBD pups, indicating that perinatal lethality of the Gcn1ΔRWDBD embryos was due to simple growth retardation. Similar to the yeast Gcn2/Gcn1 system, AAS- or UV irradiation-induced elF2α phosphorylation was diminished in the Gcn1ΔRWDBD mouse embryonic fibroblasts (MEFs), suggesting that GCN1 RWDBD is responsible for GCN2 activity. In addition, we found reduced cell proliferation and G2/M arrest accompanying a decrease in Cdk1 and Cyclin B1 in the Gcn1ΔRWDBD MEFs. Our results demonstrated, for the first time, that GCN1 is essential for both GCN2-dependent stress response and GCN2-independent cell cycle regulation.
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http://dx.doi.org/10.1371/journal.pgen.1008693DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7179835PMC
April 2020

Regulation of Nrf2 by Mitochondrial Reactive Oxygen Species in Physiology and Pathology.

Biomolecules 2020 02 17;10(2). Epub 2020 Feb 17.

Department of Stress Response Science, Center for Advanced Medical Research, Hirosaki University Graduate School of Medicine, 5 Zaifu-cho, Hirosaki 036-8562, Japan.

Reactive oxygen species (ROS) are byproducts of aerobic respiration and signaling molecules that control various cellular functions. Nrf2 governs the gene expression of endogenous antioxidant synthesis and ROS-eliminating enzymes in response to various electrophilic compounds that inactivate the negative regulator Keap1. Accumulating evidence has shown that mitochondrial ROS (mtROS) activate Nrf2, often mediated by certain protein kinases, and induce the expression of antioxidant genes and genes involved in mitochondrial quality/quantity control. Mild physiological stress, such as caloric restriction and exercise, elicits beneficial effects through a process known as "mitohormesis." Exercise induces NOX4 expression in the heart, which activates Nrf2 and increases endurance capacity. Mice transiently depleted of SOD2 or overexpressing skeletal muscle-specific UCP1 exhibit Nrf2-mediated antioxidant gene expression and PGC1α-mediated mitochondrial biogenesis. ATF4 activation may induce a transcriptional program that enhances NADPH synthesis in the mitochondria and might cooperate with the Nrf2 antioxidant system. In response to severe oxidative stress, Nrf2 induces Klf9 expression, which represses mtROS-eliminating enzymes to enhance cell death. Nrf2 is inactivated in certain pathological conditions, such as diabetes, but Keap1 down-regulation or mtROS elimination rescues Nrf2 expression and improves the pathology. These reports aid us in understanding the roles of Nrf2 in pathophysiological alterations involving mtROS.
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http://dx.doi.org/10.3390/biom10020320DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7072240PMC
February 2020

Role of Nrf2 in inflammatory response in lung of mice exposed to zinc oxide nanoparticles.

Part Fibre Toxicol 2019 12 16;16(1):47. Epub 2019 Dec 16.

Department of Occupational and Environmental Health, Nagoya University Graduate School of Medicine, Nagoya, Japan.

Background: Zinc oxide nanoparticles (ZnO-NPs) are widely used in many industrial sectors and previous studies have reported that exposure of the lungs to ZnO-NPs induces both acute and/or chronic pulmonary inflammation, but the exact mechanism underlying such response remains elusive. This study investigated the role of nuclear factor-erythroid 2-related factor (Nrf2) in pulmonary inflammation induced by exposure to ZnO-NPs using Nrf2 null (Nrf2) mice.

Methods: Twenty-four male Nrf2 mice and thirty male wild type C57BL/6 J mice were divided into three groups of eight and ten each respectively, and exposed once to ZnO-NPs at 0, 10, 30 μg/mouse by pharyngeal aspiration. At 14 days after the exposure to ZnO-NPs, bronchoalveolar lavage fluid (BALF) and lungs were collected to quantify protein level and the number of inflammatory cells. The mRNA levels of Nrf2-dependent antioxidant enzymes and inflammatory cytokines in lung tissue were measured.

Results: Exposure to ZnO-NPs dose-dependently increased the number of total cells, macrophages, lymphocytes and eosinophils in BALF both in Nrf2 mice and wild type mice, but the magnitude of increase was significantly higher in Nrf2 mice than wild type mice. The number of neutrophils in BALF increased in Nrf2 mice, being accompanied by marginal trend of increase in mRNA expression of MIP-2, neutrophil chemoattractant, but such changes were not observed in wild type mice. Exposure to ZnO-NPs did not dose-dependently increase mRNA level of Nrf2-dependent antioxidant enzymes both in Nrf2 mice and wild type mice.

Conclusion: Pharyngeal aspiration of ZnO-NPs induced infiltration of inflammatory cells in the lung of mice, but minimally induced Nrf2-dependent antioxidant enzymes. The results suggest that Nrf2 play a role in negative regulation on ZnO-NP exposure-induced neutrophil migration, but does not demonstrate that the regulation is through suppression of oxidative stress.
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http://dx.doi.org/10.1186/s12989-019-0328-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6915997PMC
December 2019

Emerging evidence for crosstalk between Nrf2 and mitochondria in physiological homeostasis and in heart disease.

Arch Pharm Res 2020 Mar 11;43(3):286-296. Epub 2019 Nov 11.

Department of Stress Response Science, Center for Advanced Medical Research, Hirosaki University Graduate School of Medicine, 5 Zaifu-cho, Hirosaki, 036-8562, Japan.

Nrf2 regulates redox homeostasis in cells by coordinately regulating a range of antioxidant enzymes and proteins. An increase in oxidative stress is one of the hallmarks of aging, and Nrf2 protein levels and activity decrease with aging. Decreased mitochondrial functions, such as decreased ATP production, also occur with aging, leading to the increased generation of reactive oxygen species (ROS) and oxidative stress. Thus, understanding the relationships between Nrf2 and the mitochondria is important for clarifying the regulatory mechanisms of aging. It is becoming clear that Nrf2 is activated in a tissue-specific manner in response to mitochondrial or NADPH oxidase-generated ROS. As the heart consists of postmitotic cells that utilize ATP produced mainly by mitochondrial oxidative phosphorylation, cardiomyocytes are equipped with highly sophisticated mitochondrial quality control mechanisms. Consistent with these findings, it has been reported that Nrf2 in the heart is regulated via a specific translational mechanism and that Nrf2 activation confers cardioprotective effects in various disease models. Thus, Nrf2 is a promising target for anti-aging strategies to combat age-related heart diseases, such as age-related cardiomyopathy.
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http://dx.doi.org/10.1007/s12272-019-01188-zDOI Listing
March 2020

Concomitant Nrf2- and ATF4-activation by Carnosic Acid Cooperatively Induces Expression of Cytoprotective Genes.

Int J Mol Sci 2019 Apr 5;20(7). Epub 2019 Apr 5.

Department of Stress Response Science, Center for Advanced Medical Research, Hirosaki University Graduate School of Medicine, Hirosaki 036-8562, Japan.

Carnosic acid (CA) is a phytochemical found in some dietary herbs, such as L., and possesses antioxidative and anti-microbial properties. We previously demonstrated that CA functions as an activator of nuclear factor, erythroid 2 (NF-E2)-related factor 2 (Nrf2), an oxidative stress-responsive transcription factor in human and rodent cells. CA enhances the expression of nerve growth factor (NGF) and antioxidant genes, such as in an Nrf2-dependent manner in U373MG human astrocytoma cells. However, CA also induces gene expression in an Nrf2-independent manner, since 50 μM of CA administration showed striking gene induction compared with the classical Nrf2 inducer -butylhydroquinone (tBHQ) in U373MG cells. By comparative transcriptome analysis, we found that CA activates activating transcription factor 4 (ATF4) in addition to Nrf2 at high doses. CA activated ATF4 in phospho-eIF2α- and heme-regulated inhibitor kinase (HRI)-dependent manners, indicating that CA activates ATF4 through the integrated stress response (ISR) pathway. Furthermore, CA activated Nrf2 and ATF4 cooperatively enhanced the expression of and many antioxidant genes while acting independently to certain client genes. Taken together, these results represent a novel mechanism of CA-mediated gene regulation evoked by Nrf2 and ATF4 cooperation.
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http://dx.doi.org/10.3390/ijms20071706DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6480217PMC
April 2019

Role of the ISR-ATF4 pathway and its cross talk with Nrf2 in mitochondrial quality control.

J Clin Biochem Nutr 2019 Jan 15;64(1):1-12. Epub 2018 Sep 15.

Department of Stress Response Science, Center for Advanced Medical Research, Hirosaki University Graduate School of Medicine, 5 Zaifu-cho, Hirosaki 036-8562, Japan.

Recent investigations have clarified the importance of mitochondria in various age-related degenerative diseases, including late-onset Alzheimer's disease and Parkinson's disease. Although mitochondrial disturbances can be involved in every step of disease progression, several observations have demonstrated that a subtle mitochondrial functional disturbance is observed preceding the actual appearance of pathophysiological alterations and can be the target of early therapeutic intervention. The signals from damaged mitochondria are transferred to the nucleus, leading to the altered expression of nuclear-encoded genes, which includes mitochondrial proteins (i.e., mitochondrial retrograde signaling). Mitochondrial retrograde signaling improves mitochondrial perturbation (i.e., mitohormesis) and is considered a homeostatic stress response against intrinsic (ex. aging or pathological mutations) and extrinsic (ex. chemicals and pathogens) stimuli. There are several branches of the mitochondrial retrograde signaling, including mitochondrial unfolded protein response (UPR), but recent observations increasingly show the importance of the ISR-ATF4 pathway in mitochondrial retrograde signaling. Furthermore, Nrf2, a master regulator of the oxidative stress response, interacts with ATF4 and cooperatively upregulates a battery of antioxidant and antiapoptotic genes while repressing the ATF4-mediated proapoptotic gene, CHOP. In this review article, we summarized the upstream and downstream mechanisms of ATF4 activation during mitochondrial stresses and disturbances and discuss therapeutic intervention against degenerative diseases by using Nrf2 activators.
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http://dx.doi.org/10.3164/jcbn.18-37DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6348405PMC
January 2019

Increase of Tumor Infiltrating γδ T-cells in Pancreatic Ductal Adenocarcinoma Through Remodeling of the Extracellular Matrix by a Hyaluronan Synthesis Suppressor, 4-Methylumbelliferone.

Pancreas 2019 02;48(2):292-298

From the Departments of Gastroenterological Surgery.

Objectives: Desmoplastic changes of extracellular matrix (ECM) containing large amounts of hyaluronan (HA) are of interest in chemo- and immunoresistance of pancreatic ductal adenocarcinoma (PDAC). The goal of this study was to evaluate the effects of 4-methylumbelliferone (MU), a selective inhibitor of HA, on ECM and to examine how MU affects adoptive immunotherapy.

Methods: The effect of MU on cell proliferation, HA synthesis and formation of ECM were investigated in four PDAC cell lines. In addition, the cytotoxicity of γδ T-cell-rich peripheral blood mononuclear cells (PBMCs) collected from healthy donors and stimulated with zoledronate and interleukin-2 was examined in the presence of MU. The amount of HA and tumor-infiltrating lymphocytes were also investigated in mice xenograft models.

Results: In vitro, 1.0 mM MU inhibited cell proliferation by 45-70% and HA synthesis by 55-80% in all four PDAC cell lines, and enhanced γδ T-cell-rich PBMC-mediated cytotoxicity against PDAC cells. In vivo, MU reduced intratumoral HA and promoted infiltration of inoculated γδ T-cells into tumor tissue, and consequently suppressed tumor growth.

Conclusions: 4-methylumbelliferone may be an effective immunosensitizer against PDAC through induction of structural changes in the ECM.
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http://dx.doi.org/10.1097/MPA.0000000000001211DOI Listing
February 2019

Emerging Regulatory Role of Nrf2 in Iron, Heme, and Hemoglobin Metabolism in Physiology and Disease.

Front Vet Sci 2018 10;5:242. Epub 2018 Oct 10.

Department of Stress Response Science, Center for Advanced Medical Research, Hirosaki University Graduate School of Medicine, Hirosaki, Japan.

Iron has played an important role in energy production since the beginning of life, as iron-catalyzed redox reactions are required for energy production. Oxygen, a highly efficient electron acceptor with high reduction potential, facilitates highly efficient energy production in eukaryotic cells. However, the increasing atmospheric oxygen concentration produces new threats to the organism, as oxygen reacts with iron and produces reactive oxygen species unless its levels are strictly regulated. As the size of multicellular organisms increases, these organisms must transport oxygen to the peripheral tissues and begin to employ red blood cells containing hemoglobin. This system is potentially a double-edged sword, as hemoglobin autoxidation occurs at a certain speed and releases free iron into the cytoplasm. Nrf2 belongs to the CNC transcription factor family, in which NF-E2p45 is the founding member. NF-E2p45 was first identified as a transcription factor that binds to the erythroid gene regulatory element NF-E2 located in the promoter region of the heme biosynthetic porphobilinogen deaminase gene. Human Nrf2 was also identified as a transcription factor that binds to the regulatory region of the β-globin gene. Despite these original findings, NF-E2p45 and Nrf2 knockout mice exhibit few erythroid phenotypes. Nrf2 regulates the expression of a wide range of antioxidant and detoxification enzymes. In this review article, we describe and discuss the roles of Nrf2 in various iron-mediated bioreactions and its possible coevolution with iron and oxygen.
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http://dx.doi.org/10.3389/fvets.2018.00242DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6191506PMC
October 2018

Aging and are determinative factors of plasma A42 levels.

Ann Clin Transl Neurol 2018 Oct 7;5(10):1184-1191. Epub 2018 Sep 7.

Department of Neurology Hrosaki University Graduate School of Medicine 5 Zaifu-cho Hirosaki 037-8562 Japan.

Objective: The aim of this study was to confirm determinative factors for plasma A and its association with cognitive function.

Methods: Fasting plasma A40 and A42 levels were measured by ELISA in 1019 participants in the Iwaki Health Promotion Project. The relationships between plasma A and health-related items, including physical characteristics, cognitive function tests, blood chemistry, and genotype were analyzed.

Results: The plasma levels of A40 and A42, and A40/42 ratio were found to significantly increase with aging. The age-dependent increase in A42 level was significantly suppressed by . Renal function was an associated factor for the plasma A40 level. The plasma A42 level and A40/42 ratio correlated with cognitive function.

Interpretation: Age and are major determinative factors of plasma levels of A42 and the A40/42 ratio. These factors are critical adjustment factors for the usage of plasma A as a biomarker of central nervous system amyloidosis.
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http://dx.doi.org/10.1002/acn3.635DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6186936PMC
October 2018

C151 in KEAP1 is the main cysteine sensor for the cyanoenone class of NRF2 activators, irrespective of molecular size or shape.

Sci Rep 2018 05 23;8(1):8037. Epub 2018 May 23.

Jacqui Wood Cancer Centre, Division of Cancer Research, School of Medicine, University of Dundee, Dundee, Scotland, United Kingdom.

Numerous small molecules (termed inducers), many of which are electrophiles, upregulate cytoprotective responses and inhibit pro-inflammatory pathways by activating nuclear factor-erythroid 2 p45-related factor 2 (NRF2). Key to NRF2 activation is the ability to chemically modifying critical sensor cysteines in the main negative regulator of NRF2, Kelch-like ECH-associated protein 1 (KEAP1), of which C151, C273 and C288 are best characterized. This study aimed to establish the requirement for these cysteine sensor(s) for the biological activities of the most potent NRF2 activators known to date, the cyclic cyanoenones, some of which are in clinical trials. It was found that C151 in KEAP1 is the main cysteine sensor for this class of inducers, irrespective of molecular size or shape. Furthermore, in primary macrophage cells expressing C151S mutant KEAP1, at low concentrations, the tricyclic cyanoenone TBE-31 is inactive as an activator of NRF2 as well as an inhibitor of lipopolysaccharide-stimulated gene expression of the pro-inflammatory cytokines IL6 and IL1β. However, at high inducer concentrations, NRF2 activation proceeds in the absence of C151, albeit at a lower magnitude. Our findings highlight the intrinsic flexibility of KEAP1 and emphasize the critical importance of establishing the precise dose of NRF2 activators for maintaining on-target selectivity.
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http://dx.doi.org/10.1038/s41598-018-26269-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5966396PMC
May 2018

Increase in proapoptotic activity of inhibitory PAS domain protein via phosphorylation by MK2.

FEBS J 2017 12 13;284(23):4115-4127. Epub 2017 Nov 13.

Division of Biomedical Measurements and Diagnostics, Graduate School of Biomedical Engineering, Tohoku University, Sendai, Japan.

Inhibitory PAS domain protein (IPAS) is a bifunctional protein that downregulates hypoxic gene expression and exerts proapoptotic activity by preventing prosurvival activity of Bcl-x and its related factors. Proapoptotic activity of IPAS is attenuated by the activation of the PINK1-Parkin pathway, and involved in neuronal degeneration in an experimental mouse model of Parkinson's disease. The current study shows that phosphorylation of IPAS at Ser184 by MAPK-activated protein kinase 2 (MK2 or MAPKAPK2) enhances the proapoptotic function of IPAS. Perinuclear clustering of mitochondria and activation of caspase-3 caused by the transient expression of EGFP-IPAS were increased by UVB irradiation. The C-terminal region of IPAS mediated the UVB susceptibility of IPAS. Increase in IPAS-induced mitochondrial clustering by UVB was completly inhibited by the p38 MAPK inhibitor SB203580. Mass spectrometry analysis of UVB-activated IPAS identified several phosphorylation sites in the C-terminal region containing p38 MAPK consensus phosphorylation sites at Ser219 and Ser223, and an MK2 consensus site at Ser184. Although mutations of Ser219 and Ser223 to Ala did not suppress the UVB-induced mitochondrial clustering, replacement of Ser184 with Ala blocked it. A phosphomimetic substitution at Ser184 enhanced mitochondrial clustering and activation of caspase-3 without UVB exposure. Furthermore, binding affinity to Bcl-x was increased by the mutation. Treatment of PC12 cells with CoCl caused activation of MK2 and mitochondrial clustering. IPAS-dependent cell death induced by CoCl in PC12 cells was decreased by the treatment with the MK2 inhibitor MK2 inhibitor III and by siRNA-directed silencing of MK2.
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http://dx.doi.org/10.1111/febs.14300DOI Listing
December 2017

Novel roles of glycosaminoglycans in the degradation of type I collagen by cathepsin K.

Glycobiology 2017 12;27(12):1089-1098

Department of Glycotechnology.

Glycosaminoglycans (GAGs) and collagen are the major organic components of bone matrix. However, their roles and functional relationships remain elusive. To investigate the role of GAGs in bone matrix degradation, the effects of GAGs on collagen were examined under acidic conditions that recapitulate the microenvironment of osteoclast resorption pits. We found that sulfated GAGs protect collagen fibrils against acid denaturation. Scanning electron microscopy demonstrated that collagen fibrils retain the fibril structure at pH 4.0 in the presence of chondroitin 6-sulfate. By surface plasmon resonance analysis, we found that sulfated GAGs, but not non-sulfated GAGs, bind to triple-helix type I collagen below pH 4.5. The binding of collagen in an acidic solution was dependent upon the GAG sugar chain length. Functionally, the acid-resistant collagen fibrils generated in the presence of sulfated GAGs were resistant to cathepsin K degradation in vitro below pH 4.0. As the pH increased from 4.0 to 5.0, the acid-resistant collagen fibrils were degraded by cathepsin K. Our results highlight the possibility that the interaction between GAGs and collagen under acidic conditions has a regulatory impact on cathepsin K-mediated bone degradation.
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http://dx.doi.org/10.1093/glycob/cwx083DOI Listing
December 2017

Effects of deficiency of Kelch-like ECH-associated protein 1 on skeletal organization: a mechanism for diminished nuclear factor of activated T cells cytoplasmic 1 during osteoclastogenesis.

FASEB J 2017 09 17;31(9):4011-4022. Epub 2017 May 17.

Division of Dental Pharmacology, Department of Developmental and Reconstructive Medicine, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan.

Kelch-like ECH-associated protein 1 (Keap1) binds to nuclear factor E2 p45-related factor 2 (Nrf2), a transcription factor for antioxidant enzymes, to suppress Nrf2 activation. The role of oxidative stress in many diseases supports the possibility that processes that are associated with Nrf2 activation might offer therapeutic potential. Nrf2 deficiency induces osteoclastogenesis, which is responsible for bone loss, by activating receptor activator of NF-κB ligand (RANKL)-mediated signaling; however, the effects of Keap1 deficiency remain unclear. By using Keap1-deficient newborn mice, we observed that talus and calcaneus bone formation was partially retarded and that osteoclast number was reduced without severe gross abnormalities. In addition, Keap1-deficient macrophages were unable to differentiate into osteoclasts attenuation of RANKL-mediated signaling and expression of nuclear factor of activated T cells cytoplasmic 1 (NFATc1), a key transcription factor that is involved in osteoclastogenesis. Furthermore, Keap1 deficiency up-regulated the expression of , a negative regulator of NFATc1. RANKL-induced mitochondrial gene expression is required for down-regulation of IFN regulatory factor 8 (IRF-8), a negative transcriptional regulator of NFATc1. Our results indicate that Keap1 deficiency down-regulated peroxisome proliferator-activated receptor-γ coactivator 1β and mitochondrial gene expression and up-regulated expression. These results suggest that the Keap1/Nrf2 axis plays a critical role in NFATc1 expression and osteoclastogenic progression.-Sakai, E., Morita, M., Ohuchi, M., Kido, M. A., Fukuma, Y., Nishishita, K., Okamoto, K., Itoh, K., Yamamoto, M., Tsukuba, T. Effects of deficiency of Kelch-like ECH-associated protein 1 on skeletal organization: a mechanism for diminished nuclear factor of activated T cells cytoplasmic 1 during osteoclastogenesis.
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http://dx.doi.org/10.1096/fj.201700177RDOI Listing
September 2017

The BET bromodomain inhibitor exerts the most potent synergistic anticancer effects with quinone-containing compounds and anti-microtubule drugs.

Oncotarget 2016 Nov;7(48):79217-79232

Children's Cancer Institute Australia for Medical Research, University of New South Wales, Sydney, Australia.

BET bromodomain inhibitors are very promising novel anticancer agents, however, single therapy does not cause tumor regression in mice, suggesting the need for combination therapy. After screening a library of 2697 small molecule compounds, we found that two classes of compounds, the quinone-containing compounds such as nanaomycin and anti-microtubule drugs such as vincristine, exerted the best synergistic anticancer effects with the BET bromodomain inhibitor JQ1 in neuroblastoma cells. Mechanistically, the quinone-containing compound nanaomycin induced neuroblastoma cell death but also activated the Nrf2-antioxidant signaling pathway, and the BET bromodomain proteins BRD3 and BRD4 formed a protein complex with Nrf2. Treatment with JQ1 blocked the recruitment of Nrf2 to the antioxidant responsive elements at Nrf2 target gene promoters, and JQ1 exerted synergistic anticancer effects with nanaomycin by blocking the Nrf2-antioxidant signaling pathway. JQ1 and vincristine synergistically induced neuroblastoma cell cycle arrest at the G2/M phase, aberrant mitotic spindle assembly formation and apoptosis, but showed no effect on cell survival in normal non-malignant cells. Importantly, co-treatment with JQ1 and vincristine synergistically suppressed tumor progression in neuroblastoma-bearing mice. These results strongly suggest that patients treated with BET bromodomain inhibitors in clinical trials should be co-treated with vincristine.
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http://dx.doi.org/10.18632/oncotarget.12640DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5346709PMC
November 2016

The role of NUB1 in α-synuclein degradation in Lewy body disease model mice.

Biochem Biophys Res Commun 2016 Feb 18;470(3):635-642. Epub 2016 Jan 18.

Department of Neuropathology, Institute of Brain Science, Hirosaki University Graduate School of Medicine, 5 Zaifu-cho, Hirosaki 036-8562, Japan.

Abnormal α-synuclein is deposited in neuronal cytoplasmic inclusions and presynapses in Parkinson's disease (PD) and dementia with Lewy bodies (DLB). Previously we have shown that NUB1 is accumulated in these specific regions together with abnormal α-synuclein and that NUB1 is able to inhibit α-synuclein aggregation in cultured cells. We therefore created transgenic (Tg) mice expressing both NUB1 and abnormal α-synuclein to investigate the role of NUB1 on degradation of abnormal α-synuclein in vivo. Immunohistochemical and biochemical studies confirmed that NUB1 was over-expressed in neurons of mice expressing NUB1 (NUB1 Tg), and both NUB1 and abnormal α-synuclein (double Tg). NUB1 levels were increased by 4.7-fold in NUB1 Tg mice compared with wild type mice. Unexpectedly, normal and abnormal α-synuclein levels were unchanged between abnormal α-synuclein Tg mice (Lewy body disease model mice) and double Tg mice, and pathological observations were almost similar between them. Finally, we found that the levels of insoluble α-synuclein were lower and those of some chaperone molecules were higher in double Tg mice compared with abnormal α-synuclein Tg mice. These results suggest that increased levels of NUB1 play a potential role in degradation of detergent-insoluble α-synuclein in vivo, although it is insufficient to degrade abnormal α-synuclein in Lewy body disease model mice.
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http://dx.doi.org/10.1016/j.bbrc.2016.01.093DOI Listing
February 2016

Trehalose intake induces chaperone molecules along with autophagy in a mouse model of Lewy body disease.

Biochem Biophys Res Commun 2015 Oct 20;465(4):746-52. Epub 2015 Aug 20.

Department of Neuropathology, Institute of Brain Science, Hirosaki University Graduate School of Medicine, 5 Zaifu-cho, Hirosaki 036-8562, Japan.

The accumulation of mis-folded and/or abnormally modified proteins is a major characteristic of many neurodegenerative diseases. In Lewy body disease (LBD), which includes Parkinson's disease and dementia with Lewy bodies, insoluble α-synuclein is widely deposited in the presynaptic terminals as well as in the neuronal cytoplasm in distinct brain regions. It is well known that the autophagy-lysosome system serves as an efficient degradation pathway for abnormal molecules within cells. To test the possibility that activated autophagy can degrade abnormal molecules, we investigated the effect of trehalose on abnormal aggregation of α-synuclein in a model of LBD. Trehalose is a natural disaccharide composed of two glucose units and functions as an autophagy inducer. Consistent with previous studies, trehalose increased level of the autophagosomal protein LC3, especially a lipidated form LC3-II in cultured cells and mice brain. Also, trehalose increased levels of several chaperon molecules, such as HSP90 and SigmaR1, in the brains of LBD model mice. Further studies revealed that level of detergent-insoluble α-synuclein was suppressed in mice following oral administration of trehalose, despite an apparent alteration was not observed regarding abnormal aggregation of α-synuclein. These results suggest that the oral intake of trehalose modulates propensity of molecules prior to aggregation formation.
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http://dx.doi.org/10.1016/j.bbrc.2015.08.076DOI Listing
October 2015
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