Publications by authors named "Norihiko Takeda"

125 Publications

Uterine Epithelial LIF Receptors Contribute to Implantation Chamber Formation in Blastocyst Attachment.

Endocrinology 2021 Nov;162(11)

Department of Obstetrics and Gynecology, Graduate School of Medicine, The University of Tokyo, Tokyo 113-8655, Japan.

Recent studies have demonstrated that the formation of an implantation chamber composed of a uterine crypt, an implantation-competent blastocyst, and uterine glands is a critical step in blastocyst implantation in mice. Leukemia inhibitory factor (LIF) activates signal transducer and activator of transcription 3 (STAT3) precursors via uterine LIF receptors (LIFRs), allowing successful blastocyst implantation. Our recent study revealed that the role of epithelial STAT3 is different from that of stromal STAT3. However, both are essential for blastocyst attachment, suggesting the different roles of epithelial and stromal LIFR in blastocyst implantation. However, how epithelial and stromal LIFR regulate the blastocyst implantation process remains unclear. To investigate the roles of LIFR in the uterine epithelium and stroma, we generated Lifr-floxed/lactoferrin (Ltf)-iCre (Lifr eKO) and Lifr-floxed/antimüllerian hormone receptor type 2 (Amhr2)-Cre (Lifr sKO) mice with deleted epithelial and stromal LIFR, respectively. Surprisingly, fertility and blastocyst implantation in the Lifr sKO mice were normal despite stromal STAT3 inactivation. In contrast, blastocyst attachment failed, and no implantation chambers were formed in the Lifr eKO mice with epithelial inactivation of STAT3. In addition, normal responsiveness to ovarian hormones was observed in the peri-implantation uteri of the Lifr eKO mice. These results indicate that the epithelial LIFR-STAT3 pathway initiates the formation of implantation chambers, leading to complete blastocyst attachment, and that stromal STAT3 regulates blastocyst attachment without stromal LIFR control. Thus, uterine epithelial LIFR is critical to implantation chamber formation and blastocyst attachment.
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http://dx.doi.org/10.1210/endocr/bqab169DOI Listing
November 2021

NK cells in hypoxic skin mediate a trade-off between wound healing and antibacterial defence.

Nat Commun 2021 08 4;12(1):4700. Epub 2021 Aug 4.

University of Zurich, Institute of Anatomy, Zurich, Switzerland.

During skin injury, immune response and repair mechanisms have to be coordinated for rapid skin regeneration and the prevention of microbial infections. Natural Killer (NK) cells infiltrate hypoxic skin lesions and Hypoxia-inducible transcription factors (HIFs) mediate adaptation to low oxygen. We demonstrate that mice lacking the Hypoxia-inducible factor (HIF)-1α isoform in NK cells show impaired release of the cytokines Interferon (IFN)-γ and Granulocyte Macrophage - Colony Stimulating Factor (GM-CSF) as part of a blunted immune response. This accelerates skin angiogenesis and wound healing. Despite rapid wound closure, bactericidal activity and the ability to restrict systemic bacterial infection are impaired. Conversely, forced activation of the HIF pathway supports cytokine release and NK cell-mediated antibacterial defence including direct killing of bacteria by NK cells despite delayed wound closure. Our results identify, HIF-1α in NK cells as a nexus that balances antimicrobial defence versus global repair in the skin.
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http://dx.doi.org/10.1038/s41467-021-25065-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8338923PMC
August 2021

Hyperpolarized C Magnetic Resonance Imaging as a Tool for Imaging Tissue Redox State, Oxidative Stress, Inflammation, and Cellular Metabolism.

Antioxid Redox Signal 2021 Aug 17. Epub 2021 Aug 17.

Division of Bioengineering & Bioinformatics, Graduate School of Information Science & Technology, Hokkaido University, Sapporo, Japan.

Magnetic resonance imaging (MRI) with hyperpolarized (HP) C-labeled redox-sensitive metabolic tracers can provide noninvasive functional imaging biomarkers, reflecting tissue redox state, oxidative stress, and inflammation, among others. The capability to use endogenous metabolites as C-enriched imaging tracers without structural modification makes HP C MRI a promising tool to evaluate redox state in patients with various diseases. Recent studies have demonstrated the feasibility of metabolic imaging of C-labeled tracers polarized by parahydrogen-induced polarization techniques, which offer a cost-effective alternative to the more widely used dissolution dynamic nuclear polarization-based hyperpolarizers. Although the fluxes of many metabolic pathways reflect the change in tissue redox state, they are not functionally specific. In the present review, we summarize recent challenges in the development of specific C metabolic tracers for biomarkers of redox state, including that for detecting reactive oxygen species. Applications of HP C metabolic MRI to evaluate redox state have only just begun to be investigated. The possibility to gain a comprehensive understanding of the correlations between tissue redox potential and metabolism under different pathological conditions by using HP C MRI is promoting its interest in the clinical arena, along with its noninvasive biomarkers to evaluate the extent of disease and treatment response.
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http://dx.doi.org/10.1089/ars.2021.0139DOI Listing
August 2021

Hyperpolarized C Magnetic Resonance Imaging of Fumarate Metabolism by Parahydrogen-induced Polarization: A Proof-of-Concept in vivo Study.

Chemphyschem 2021 05;22(10):905

Division of Bioengineering & Bioinformatics, Graduate School of Information Science & Technology, Hokkaido University, North 14, West 9, Kita-ku, Sapporo, Hokkaido, 060-0814, Japan.

The front cover artwork is provided by the group of Dr. Neil J. Stewart, Prof. Hiroshi Hirata, and Dr. Shingo Matsumoto (Hokkaido University, Japan) as well as Dr. Takuya Hashimoto (Chiba University, Japan). The image shows hyperpolarized C fumarate metabolism to hyperpolarized C malate, which is released into the extracellular space in regions of necrotic cell death, where the cell membrane is disrupted. Read the full text of the Article at 10.1002/cphc.202001038.
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http://dx.doi.org/10.1002/cphc.202100338DOI Listing
May 2021

Mutant KRAS drives metabolic reprogramming and autophagic flux in premalignant pancreatic cells.

Cancer Gene Ther 2021 Apr 8. Epub 2021 Apr 8.

Department of Gastroenterology, Graduate School of Medicine, The University of Tokyo, Tokyo, 113-8655, Japan.

Mutational activation of the KRAS gene occurs in almost all pancreatic ductal adenocarcinoma (PDAC) and is the earliest molecular event in their carcinogenesis. Evidence has accumulated of the metabolic reprogramming in PDAC, such as amino acid homeostasis and autophagic flux. However, the biological effects of KRAS mutation on metabolic reprogramming at the earlier stages of PDAC carcinogenesis are unclear. Here we report dynamic metabolic reprogramming in immortalized human non-cancerous pancreatic ductal epithelial cells, in which a KRAS mutation was induced by gene-editing, which may mimic early pancreatic carcinogenesis. Similar to the cases of PDAC, KRAS gene mutation increased the dependency on glucose and glutamine for maintaining the intracellular redox balance. In addition, the intracellular levels of amino acids were significantly decreased because of active protein synthesis, and the cells required greater autophagic flux to maintain their viability. The lysosomal inhibitor chloroquine significantly inhibited cell proliferation. Therefore, metabolic reprogramming is an early event in carcinogenesis initiated by KRAS gene mutation, suggesting a rationale for the development of nutritional interventions that suppress or delay the development of PDAC.
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http://dx.doi.org/10.1038/s41417-021-00326-4DOI Listing
April 2021

Establishment of Novel Protein Interaction Assays between Sin3 and REST Using Surface Plasmon Resonance and Time-Resolved Fluorescence Energy Transfer.

Int J Mol Sci 2021 Feb 26;22(5). Epub 2021 Feb 26.

Center for Therapeutic Innovation, Gene Research Center for Frontiers Life Sciences, Nagasaki University, Graduate School of Biomedical Sciences, 1-12-14 Sakamoto, Nagasaki 852-8523, Japan.

Repressor element-1 (RE-1) or neural restrictive silencer element (NRSE) bound with a zinc finger transcription repressor, RE-1 silencing transcription factor (REST, also known as neural restrictive silencer factor, NRSF) has been identified as a fundamental repressor element in many genes, including neuronal genes. Genes regulated by REST/NRSF regulate multifaceted neuronal phenotypes, and their defects in the machinery cause neuropathies, disorders of neuron activity), autism and so on. In REST repressions, the N-terminal repressor domain recruits Sin3B via its paired amphipathic helix 1 (PAH1) domain, which plays an important role as a scaffold for histone deacetylase 1 and 2. This machinery has a critical role in maintaining neuronal robustness. In this study, in order to establish protein-protein interaction assays mimicking a binding surface between Sin3B and REST, we selected important amino acids from structural information of the PAH1/REST complex and then tried to reconstitute it using recombinant short peptides derived from PAH1/REST. Initially, we validated whether biotinylated REST interacts with glutathione S-transferase (GST)-tagged PAH1 and whether another PAH1 peptide (PAH1-FLAG) competitively binds with biotinylated REST using surface plasmon resonance (SPR). We observed a direct interaction and competitive binding of two PAH1 peptides. Secondly, in order to establish a high-throughput and high-dynamic-range assay, we utilized an easily performed novel time-resolved fluorescence energy transfer (TR-FRET) assay, and closely monitored this interaction. Finally, we succeeded in establishing a novel high-quality TR-FRET assay and a novel interaction assay based on SPR.
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http://dx.doi.org/10.3390/ijms22052323DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7956749PMC
February 2021

Hyperpolarized C Magnetic Resonance Imaging of Fumarate Metabolism by Parahydrogen-induced Polarization: A Proof-of-Concept in vivo Study.

Chemphyschem 2021 05 18;22(10):915-923. Epub 2021 Mar 18.

Division of Bioengineering & Bioinformatics, Graduate School of Information Science & Technology, Hokkaido University, North 14, West 9, Kita-ku, Sapporo, Hokkaido, 060-0814, Japan.

Hyperpolarized [1- C]fumarate is a promising magnetic resonance imaging (MRI) biomarker for cellular necrosis, which plays an important role in various disease and cancerous pathological processes. To demonstrate the feasibility of MRI of [1- C]fumarate metabolism using parahydrogen-induced polarization (PHIP), a low-cost alternative to dissolution dynamic nuclear polarization (dDNP), a cost-effective and high-yield synthetic pathway of hydrogenation precursor [1- C]acetylenedicarboxylate (ADC) was developed. The trans-selectivity of the hydrogenation reaction of ADC using a ruthenium-based catalyst was elucidated employing density functional theory (DFT) simulations. A simple PHIP set-up was used to generate hyperpolarized [1- C]fumarate at sufficient C polarization for ex vivo detection of hyperpolarized C malate metabolized from fumarate in murine liver tissue homogenates, and in vivo C MR spectroscopy and imaging in a murine model of acetaminophen-induced hepatitis.
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http://dx.doi.org/10.1002/cphc.202001038DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8251594PMC
May 2021

CXCR7 ameliorates myocardial infarction as a β-arrestin-biased receptor.

Sci Rep 2021 Feb 9;11(1):3426. Epub 2021 Feb 9.

Department of Cardiovascular Medicine, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-8655, Japan.

Most seven transmembrane receptors (7TMRs) are G protein-coupled receptors; however, some 7TMRs evoke intracellular signals through β-arrestin as a biased receptor. As several β-arrestin-biased agonists have been reported to be cardioprotective, we examined the role of the chemokine receptor CXCR7 as a β-arrestin-biased receptor in the heart. Among 510 7TMR genes examined, Cxcr7 was the most abundantly expressed in the murine heart. Single-cell RNA-sequencing analysis revealed that Cxcr7 was abundantly expressed in cardiomyocytes and fibroblasts. Cardiomyocyte-specific Cxcr7 null mice showed more prominent cardiac dilatation and dysfunction than control mice 4 weeks after myocardial infarction. In contrast, there was no difference in cardiac phenotypes between fibroblast-specific Cxcr7-knockout mice and control mice even after myocardial infarction. TC14012, a specific agonist of CXCR7, significantly recruited β-arrestin to CXCR7 in CXCR7-expressing cells and activated extracellular signal-regulated kinase (ERK) in neonatal rat cardiomyocytes. Cxcr7 expression was significantly increased and ERK was activated in the border zone of the heart in control, but not Cxcr7 null mice. These results indicate that the abundantly expressed CXCR7 in cardiomyocytes may play a protective role in the heart as a β-arrestin-biased receptor and that CXCR7 may be a novel therapeutic target for myocardial infarction.
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http://dx.doi.org/10.1038/s41598-021-83022-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7873251PMC
February 2021

Retinoblastoma protein promotes uterine epithelial cell cycle arrest and necroptosis for embryo invasion.

EMBO Rep 2021 02 5;22(2):e50927. Epub 2021 Jan 5.

Department of Obstetrics and Gynecology, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan.

Retinoblastoma protein (RB) encoded by Rb1 is a prominent inducer of cell cycle arrest (CCA). The hormone progesterone (P ) promotes CCA in the uterine epithelium and previous studies indicated that P activates RB by reducing the phosphorylated, inactive form of RB. Here, we show that embryo implantation is impaired in uterine-specific Rb1 knockout mice. We observe persistent cell proliferation of the Rb1-deficient uterine epithelium until embryo attachment, loss of epithelial necroptosis, and trophoblast phagocytosis, which correlates with subsequent embryo invasion failure, indicating that Rb1-induced CCA and necroptosis of uterine epithelium are involved in embryo invasion. Pre-implantation P supplementation is sufficient to restore these defects and embryo invasion. In Rb1-deficient uterine epithelial cells, TNFα-primed necroptosis is impaired, which is rescued by the treatment with a CCA inducer thymidine or P through the upregulation of TNF receptor type 2. TNFα is expressed in the luminal epithelium and the embryo at the embryo attachment site. These results provide evidence that uterine Rb1-induced CCA is involved in TNFα-primed epithelial necroptosis at the implantation site for successful embryo invasion.
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http://dx.doi.org/10.15252/embr.202050927DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7857437PMC
February 2021

Sequential Nucleophilic Arylation/Ring-Contractive Rearrangement of -Alkoxylactams.

Org Lett 2020 Dec 2;22(24):9740-9744. Epub 2020 Dec 2.

Kobe Pharmaceutical University, Motoyamakita, Higashinada, Kobe 658-8558, Japan.

A nucleophilic addition/ring-contractive rearrangement of α-bromo -alkoxylactams with organometallic reagents was developed, providing an efficient access to α-acylpyrrolidines incorporating various C() units such as aryl, heteroaryl, and alkenyl groups. The sequential reaction proceeds through a five-membered chelate formation using nucleophilic addition followed by ring contraction via the formation of ,-hemiaminal with good yields and a broad substrate scope. Moreover, a preliminary result with the use of the chiral -alkoxylactam for the diastereoselective reaction is described.
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http://dx.doi.org/10.1021/acs.orglett.0c03821DOI Listing
December 2020

Soluble VCAM-1 promotes gemcitabine resistance via macrophage infiltration and predicts therapeutic response in pancreatic cancer.

Sci Rep 2020 12 3;10(1):21194. Epub 2020 Dec 3.

Department of Gastroenterology, Graduate School of Medicine, The University of Tokyo, Bunkyo-ku, Tokyo, 113-8655, Japan.

Pancreatic cancer is one of the malignant diseases with the worst prognosis. Resistance to chemotherapy is a major difficulty in treating the disease. We analyzed plasma samples from a genetically engineered mouse model of pancreatic cancer and found soluble vascular cell adhesion molecule-1 (sVCAM-1) increases in response to gemcitabine treatment. VCAM-1 was expressed and secreted by murine and human pancreatic cancer cells. Subcutaneous allograft tumors with overexpression or knock-down of VCAM-1, as well as VCAM-1-blocking treatment in the spontaneous mouse model of pancreatic cancer, revealed that sVCAM-1 promotes tumor growth and resistance to gemcitabine treatment in vivo but not in vitro. By analyzing allograft tumors and co-culture experiments, we found macrophages were attracted by sVCAM-1 to the tumor microenvironment and facilitated resistance to gemcitabine in tumor cells. In a clinical setting, we found that the change of sVCAM-1 in the plasma of patients with advanced pancreatic cancer was an independent prognostic factor for gemcitabine treatment. Collectively, gemcitabine treatment increases the release of sVCAM-1 from pancreatic cancer cells, which attracts macrophages into the tumor, thereby promoting the resistance to gemcitabine treatment. sVCAM-1 may be a potent clinical biomarker and a potential target for the therapy in pancreatic cancer.
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http://dx.doi.org/10.1038/s41598-020-78320-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7713301PMC
December 2020

Synthesis of Pyrazoles Utilizing the Ambiphilic Reactivity of Hydrazones.

Org Lett 2020 12 16;22(23):9249-9252. Epub 2020 Nov 16.

Kobe Pharmaceutical University, Motoyamakita, Higashinada, Kobe 658-8558, Japan.

A Brønsted acid-mediated synthesis of pyrazoles from conjugated hydrazones through a β-protonation/nucleophilic addition/cyclization/aromatization sequence was developed. This protocol utilizing the ambiphilic reactivity of hydrazones enables not only self-condensation but also cross-condensation, affording multisubstituted pyrazoles in high yields, with a broad substrate scope. This sequential reaction proceeds under mild conditions via a simple operation. Moreover, the method can be applied to the synthesis of a nonsteroidal anti-inflammatory drug, Lonazolac.
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http://dx.doi.org/10.1021/acs.orglett.0c03465DOI Listing
December 2020

Febuxostat, a Xanthine Oxidase Inhibitor, Decreased Macrophage Matrix Metalloproteinase Expression in Hypoxia.

Biomedicines 2020 Nov 3;8(11). Epub 2020 Nov 3.

Department of Cardiovascular Medicine, Nagasaki University Graduate School of Biomedical Sciences, Sakamoto, Nagasaki 852-8501, Japan.

Macrophages in the atheroma region produce matrix metalloproteinases (MMPs) and decrease plaque stability. Tissue oxygen tension decreases in the arterial wall of the atherosclerotic region. Hypoxia inducible factor (HIF)-1α plays a critical role in the transcriptional activation of hypoxia inducible genes. However, the precise roles of HIF-1α independent pathways in hypoxic responses are largely unknown. Xanthine oxidase (XO) is an enzyme that utilizes molecular oxygen and produces reactive oxygen species (ROS). Here, we show that ROS derived from XO increases -3, -10, and -13 expression in murine macrophages. We found that the transcript levels of macrophage -3, -10, and -13 were increased in hypoxic conditions. Hypoxia induced MMP expression in HIF-1α deficient macrophages. -acetylcysteine (NAC) or febuxostat, an XO inhibitor, suppressed MMP expression in murine macrophages. Febuxostat decreased the incidence of plaque rupture in apolipoprotein-E-deficient mice. Our results indicate that febuxostat stabilized atherosclerotic plaque via suppressing the activities of macrophage MMP-9 and -13. Febuxostat administration is a potential therapeutic option in the management of atherosclerotic patients.
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http://dx.doi.org/10.3390/biomedicines8110470DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7693746PMC
November 2020

Uterine Epithelial Progesterone Receptor Governs Uterine Receptivity Through Epithelial Cell Differentiation.

Endocrinology 2020 12;161(12)

Department of Obstetrics and Gynecology, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan.

Progesterone receptor (PGR) is indispensable for pregnancy in mammals. Uterine PGR responds to the heightened levels of ovarian progesterone (P4) after ovulation and regulates uterine gene transcription for successful embryo implantation. Although epithelial and stromal P4-PGR signaling may interact with each other to form appropriate endometrial milieu for uterine receptivity and the subsequent embryo attachment, it remains unclear what the specific roles of epithelial P4-PGR signaling in the adult uterus are. Here we generated mice with epithelial deletion of Pgr in the adult uterus (Pgrfl/flLtfCre/+ mice) by crossing Pgr-floxed and Ltf-Cre mice. Pgrfl/flLtfCre/+ mice are infertile due to the impairment of embryo attachment. Pgrfl/flLtfCre/+ uteri did not exhibit epithelial growth arrest, suggesting compromised uterine receptivity. Both epithelial and stromal expressions of P4-responsive genes decreased in Pgrfl/flLtfCre/+ mice during the peri-implantation period, indicating that epithelial Pgr deletion affects not only epithelial but stromal P4 responsiveness. In addition, uterine LIF, an inducer of embryo attachment, was decreased in Pgrfl/flLtfCre/+ mice. The RNA-seq analysis using luminal epithelial specimens dissected out by laser capture microdissection revealed that the signaling pathways related to extracellular matrix, cell adhesion, and cell proliferation are altered in Pgr fl/flLtf Cre/+ mice. These findings suggest that epithelial PGR controls both epithelial and stromal P4 responsiveness and epithelial cell differentiation, which provides normal uterine receptivity and subsequent embryo attachment.
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http://dx.doi.org/10.1210/endocr/bqaa195DOI Listing
December 2020

Inverted Region in Bimolecular Electron Transfer in Solution Enabled by Delocalization.

J Am Chem Soc 2020 10 13;142(42):17997-18004. Epub 2020 Oct 13.

Chemistry Division, Brookhaven National Laboratory, Upton, New York 11937, United States.

Rate constants for bimolecular electron transfer (ET) increased with driving force, -Δ°, reached a plateau, and then decreased in an inverted region. This rate data was described well by electron transfer theory subject to a diffusion-controlled limit. These were for ET from radical anions of polydecylthiophene (P3DT) to a series of acceptors in THF solution. When the donor was the smaller anion of quaterthiophene (T) the inverted region was much less prominent and still less so for when the donor was the anion of bithiophene (T). Description of the data using ET theory identifies smaller electronic couplings for the highly delocalized P3DT anions as enabling the inverted behavior: The presence of a Marcus inverted region is a consequence of delocalized electronic states. The results further imply that electronic couplings smaller than usually found for molecules in contact could boost efficiency of energy storage by electron transfer and identifies size-mismatch as an important concept in control of electronic couplings.
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http://dx.doi.org/10.1021/jacs.0c04780DOI Listing
October 2020

Differential roles of uterine epithelial and stromal STAT3 coordinate uterine receptivity and embryo attachment.

Sci Rep 2020 09 23;10(1):15523. Epub 2020 Sep 23.

Department of Obstetrics and Gynecology, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-8655, Japan.

Although it has been reported that uterine signal transducer and activator of transcription 3 (STAT3) is essential for embryo implantation, the exact roles of uterine epithelial and stromal STAT3 on embryo implantation have not been elucidated. To address this issue, we generated Stat3-floxed/Ltf-iCre (Stat3-eKO), Stat3-floxed/Amhr2-Cre (Stat3-sKO), and Stat3-floxed/Pgr-Cre (Stat3-uKO) mice to delete Stat3 in uterine epithelium, uterine stroma, and whole uterine layers, respectively. We found that both epithelial and stromal STAT3 have critical roles in embryo attachment because all the Stat3-eKO and Stat3-sKO female mice were infertile due to implantation failure without any embryo attachment sites. Stat3-eKO uteri showed indented structure of uterine lumen, indicating the role of epithelial STAT3 in slit-like lumen formation in the peri-implantation uterus. Stat3-sKO uteri exhibited hyper-estrogenic responses and persistent cell proliferation of the epithelium in the peri-implantation uterus, suggesting the role of stromal STAT3 in uterine receptivity. In addition, Stat3-uKO female mice possessed not only the characteristic of persistent epithelial proliferation but also that of indented structure of uterine lumen. These findings indicate that epithelial STAT3 controls the formation of slit-like structure in uterine lumen and stromal STAT3 suppresses epithelial estrogenic responses and cell proliferation. Thus, epithelial and stromal STAT3 cooperatively controls uterine receptivity and embryo attachment through their different pathways.
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http://dx.doi.org/10.1038/s41598-020-72640-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7511330PMC
September 2020

Copper-Catalyzed Sequential Cyclization/Migration of Alkynyl Hydrazides for Construction of Ring-Expanded N-N Fused Pyrazolones.

Org Lett 2020 Sep 21;22(17):6852-6857. Epub 2020 Aug 21.

Kobe Pharmaceutical University, Motoyamakita, Higashinada, Kobe 658-8558, Japan.

A copper-catalyzed sequential cyclization/migration reaction of alkynyl hydrazides for the synthesis of ring-expanded N-N fused pyrazolones was developed. Control experiments indicate that the copper-ligand complex plays an essential role in the reaction. This approach features a broad scope including some functional group tolerance as well as a nucleophilic addition/1,3-migration/formal 1,2-migration sequence. This protocol provides simple manipulation and less waste due to high yield and atom economy. The synthetic utility of N-N fused pyrazolones was also demonstrated by further transformations.
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http://dx.doi.org/10.1021/acs.orglett.0c02378DOI Listing
September 2020

Reverse Remodeling and Current Medical Therapy in Heart Failure with Reduced Ejection Fraction.

Int Heart J 2020;61(2):197-198

Department of Cardiovascular Medicine, Graduate School of Medicine, The University of Tokyo.

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http://dx.doi.org/10.1536/ihj.20-034DOI Listing
June 2020

Hypoxia-inducible factor-1 alpha maintains mouse articular cartilage through suppression of NF-κB signaling.

Sci Rep 2020 03 25;10(1):5425. Epub 2020 Mar 25.

Sensory & Motor System Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-8655, Japan.

HIF-1α, an essential transcription factor under hypoxic condition, is indispensable for chondrocytes during skeletal development but its expression and roles in articular chondrocytes are yet to be revealed. We examined HIF-1α protein expression and the hypoxic condition during mouse osteoarthritis (OA) development using state of the art hypoxic probes and found that its expression decreased as OA progressed, coinciding with the change in hypoxic conditions in articular cartilage. Gain- and loss-of-function of HIF-1α in cell culture experiments showed that HIF-1α suppressed catabolic genes such as Mmp13 and Hif2a. We confirmed these anticatabolic effects by measuring glycosaminoglycan release from wild type and conditional knock-out mice femoral heads cultured ex vivo. We went on to surgically induce OA in mice with chondrocyte-specific deletion of Hif1a and found that the development of OA was exacerbated. Increased expression of catabolic factors and activation of NF-κB signalling was clearly evident in the knock-out mice. By microarray analysis, C1qtnf3 was identified as a downstream molecule of HIF-1α, and experiments showed it exerted anti-catabolic effects through suppression of NF-κB. We conclude that HIF-1α has an anti-catabolic function in the maintenance of articular cartilage through suppression of NF-κB signalling.
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http://dx.doi.org/10.1038/s41598-020-62463-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7096515PMC
March 2020

Establishment of Novel High-Standard Chemiluminescent Assay for NTPase in Two Protozoans and Its High-Throughput Screening.

Mar Drugs 2020 Mar 13;18(3). Epub 2020 Mar 13.

Center for Therapeutic Innovation, Gene Research Center for Frontiers Life Sciences, Nagasaki University, Graduate School of Biomedical Sciences, 1-12-14 Sakamoto, Nagasaki 852-8523, Japan.

is a major protozoan parasite and infects human and many other warm-blooded animals. The infection leads to Toxoplasmosis, a serious issue in AIDS patients, organ transplant recipients and pregnant women. , another type of protozoa, is closely related to Infections of the protozoa in animals also causes serious diseases such as Encephalomyelitis and Myositis-Polyradiculitis in dogs or abortion in cows. Both and have similar nucleoside triphosphate hydrolases (NTPase), NcNTPase and TgNTPase-I in and , respectively. These possibly play important roles in propagation and survival. Thus, we targeted the enzymes for drug discovery and tried to establish a novel high-standard assay by a combination of original biochemical enzyme assay and fluorescent assay to determine ADP content. We then validated whether or not it can be applied to high-throughput screening (HTS). Then, it fulfilled criterion to carry out HTS in both of the enzymes. In order to identify small molecules having inhibitory effects on the protozoan enzyme, we also performed HTS using two synthetic compound libraries and an extract library derived from marine bacteria and then, identified 19 compounds and 6 extracts. Nagasaki University collected many extracts from over 18,000 marine bacteria found in local Omura bay, and continues to compile an extensive collection of synthetic compounds from numerous drug libraries established by Japanese chemists.
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http://dx.doi.org/10.3390/md18030161DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7142789PMC
March 2020

γ-C (sp)-H bond functionalisation of α,β-unsaturated amides through an umpolung strategy.

Org Biomol Chem 2020 02;18(8):1563-1566

Kobe Pharmaceutical University, Motoyamakita, Higashinada, Kobe 658-8558, Japan.

The nucleophilic γ-phenylation and γ-alkylation of α,β-unsaturated amides have been developed. This umpolung reaction allows the regioselective introduction of phenyl and alkyl groups to a vinylketene N,O-acetal, which is generated in situ from an α,β-unsaturated N-alkoxyamide, followed by N-O bond cleavage in a two-step, one-pot process.
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http://dx.doi.org/10.1039/d0ob00125bDOI Listing
February 2020

Novel Reporter System Monitoring IL-18 Specific Signaling Can Be Applied to High-Throughput Screening.

Mar Drugs 2020 Jan 17;18(1). Epub 2020 Jan 17.

Center for Therapeutic Innovation, Gene Research Center for Frontiers Life Sciences, Nagasaki University, Graduate School of Biomedical Sciences, 1-12-14 Sakamoto, Nagasaki 852-8523-0022, Japan.

Very recently, the immunotherapies against cancer, autoimmune diseases, and infection have been feasible and promising. Thus, we have examined the possibility whether or not human gamma delta T cells can be applied for the novel immunotherapies. We previously established the cells stably maintaining NFkB-driven human secreted embryonic alkaline phosphatase (SEAP) expression. The cells can be used to determine the transcription activity of NFkB with high-standard dynamic range and accuracy. Because IL-18 is a kind of cytokines that enhances cytotoxicity and activity of human gamma delta T cells through NFkB activation, we have focused on the activity and signaling of IL-18. In this study, we modified the previous reporter cell that can determine the transcription activity of NFkB to express two subunits consisted of human IL-18 receptor. The modified cells secreted SEAP in response to treatment with human recombinant IL-18 in a concentration-dependent manner. We also observed the concentration-dependently enhancement of NFkB activity in the cells treated with mouse recombinant IL-18 although the affinity was lower compared to human recombinant IL-18. We also previously established the cells stably expressing and secreting human recombinant IL-18 and then validated whether or not the conditioned medium from the cells activate NFkB transcription activity using this assay. Our university has kept collecting many extracts from over 18,000 marine bacteria in our local sea around Omura bay-fungi, plants for Chinese herbal medicine, and so on-and also have kept gathering synthetic compounds from many Japanese chemists as drug libraries. Finally, in order to identify drugs mimicking IL-18 biological activity or possessing inhibitory effects on IL-18-induced NFkB, we demonstrated drug screening using number of extracts derived from marine bacteria and synthetic compounds.
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http://dx.doi.org/10.3390/md18010060DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7024245PMC
January 2020

[The roles of hypoxia signaling in tissue remodeling].

Authors:
Norihiko Takeda

Nihon Yakurigaku Zasshi 2020 ;155(1):26-29

Department of Cardiovascular Medicine, Graduate School of Medicine, The University of Tokyo.

A molecular oxygen is essential to keep a physiological activity of each organ or a cell. There exists a heterogeneity in a level of oxygen concentration in each organ. In addition, tissue oxygen concentration fluctuates dynamically during physiological activities or in pathological processes. A decrease in tissue oxygen concentration, termed as hypoxia, significantly influences the function in each organ or cell. For example, a transcript level in each gene tends to be reduced under hypoxic condition. On the other hand, some of the gene expressions are increased significantly in hypoxia, which are termed as hypoxia responsive genes. A group of transcription factor, hypoxia inducible factor (HIF)-1α and HIF-2α play a critical role in the transactivation processes of hypoxia responsive genes. Recently, the molecular processes have been elucidated by which hypoxic environment activates HIF-1α or HIF-2α activity. A preclinical animal model revealed that HIF-α signal plays a critical role in inflammation or tissue remodeling. While HIF-1α and HIF-2α usually work synergistically in inducing their target gene expressions, macrophage HIF-1α and HIF-2α act antagonistically with regard to the synthesis of nitric oxide, a potent inflammatory mediator. This review summarizes the current understanding on the roles of HIF-α mediated hypoxic responses in inflammation or tissue remodeling.
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http://dx.doi.org/10.1254/fpj.19114DOI Listing
January 2020

Noonan syndrome-associated biallelic LZTR1 mutations cause cardiac hypertrophy and vascular malformations in zebrafish.

Mol Genet Genomic Med 2020 03 28;8(3):e1107. Epub 2019 Dec 28.

Department of Pediatrics, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan.

Background: Variants in the LZTR1 (leucine-zipper-like transcription regulator 1) gene (OMIM #600574) have been reported in recessive Noonan syndrome patients. In vivo evidence from animal models to support its causative role is lacking.

Methods: By CRISPR-Cas9 genome editing, we generated lztr1-mutated zebrafish (Danio rerio). Analyses of histopathology and downstream signaling were performed to investigate the pathogenesis of cardiac and extracardiac abnormalities in Noonan syndrome.

Results: A frameshift deletion allele was created in the zebrafish lztr1. Crosses of heterozygotes obtained homozygous lztr1 null mutants that modeled LZTR1 loss-of-function. Histological analyses of the model revealed ventricular hypertrophy, the deleterious signature of Noonan syndrome-associated cardiomyopathy. Further, assessment for extracardiac abnormalities documented multiple vascular malformations, resembling human vascular pathology caused by RAS/MAPK activation. Due to spatiotemporal regulation of LZTR1, its downstream function was not fully elucidated from western blots of adult tissue.

Conclusion: Our novel zebrafish model phenocopied human recessive Noonan syndrome and supported the loss-of-function mechanism of disease-causing LZTR1 variants. The discovery of vascular malformations in mutants calls for the clinical follow-up of patients to monitor for its emergence. The model will serve as a novel platform for investigating the pathophysiology linking RAS/MAPK signaling to cardiac and vascular pathology.
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http://dx.doi.org/10.1002/mgg3.1107DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7057116PMC
March 2020

Neuron-derived VEGF contributes to cortical and hippocampal development independently of VEGFR1/2-mediated neurotrophism.

Dev Biol 2020 03 30;459(2):65-71. Epub 2019 Nov 30.

Department of Anatomy, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo, 160-8582, Japan. Electronic address:

Vascular endothelial growth factor (VEGF) is a potent mitogen critical for angiogenesis and organogenesis. Deletion or inhibition of VEGF during development not only profoundly suppresses vascular outgrowth, but significantly affects the development and function of various organs. In the brain, VEGF is thought to not only promote vascular growth, but also directly act on neurons as a neurotrophic factor by activating VEGF receptors. In the present study, we demonstrated that deletion of VEGF using hGfap-Cre line, which recombines genes specifically in cortical and hippocampal neurons, severely impaired brain organization and vascularization of these regions. The mutant mice had motor deficits, with lethality around the time of weaning. Multiple reporter lines indicated that VEGF was highly expressed in neurons, but that its cognate receptors, VEGFR1 and 2 were exclusive to endothelial cells in the brain. In accordance, mice lacking neuronal VEGFR1 and VEGFR2 did not exhibit neuronal deformities or lethality. Taken together, our data suggest that neuron-derived VEGF contributes to cortical and hippocampal development likely through angiogenesis independently of direct neurotrophic effects mediated by VEGFR1 and 2.
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http://dx.doi.org/10.1016/j.ydbio.2019.11.016DOI Listing
March 2020

Levonorgestrel Inhibits Embryo Attachment by Eliminating Uterine Induction of Leukemia Inhibitory Factor.

Endocrinology 2020 02;161(2)

Department of Obstetrics and Gynecology, Graduate School of Medicine, The University of Tokyo, Japan.

Progestogens including progesterone (P4) and levonorgestrel (LNG) are clinically used for multiple purposes such as contraception and infertility treatment. The effects of progestogens on the uterus remains to be elucidated. Here we examine the effect of excessive progestogen administration on embryo implantation focusing on the function of uterine leukemia inhibitory factor (LIF), a cytokine that is induced by estrogen and essential for embryo attachment. Treatment of wild-type (WT) female mice with vehicle (control), LNG at the dose of 300 μg/kg/day and P4 at the dose of 10 mg/day from day 1 to day 4 of pregnancy was conducted. LNG-treated and P4-treated mice showed embryo attachment failure on day 5 of pregnancy (The rate of mice with embryo attachment sites [%MAS], 11% and 13%, respectively), while all the control mice had normal attachment sites. Uterine LIF expression was significantly reduced in LNG-treated and P4-treated mice on day 4 evening. Administration of recombinant LIF (rLIF) at the dose of 24 μg/day on day 4 significantly rescued embryo attachment failure in LNG-treated and P4-treated mice (%MAS, 80% and 75%, respectively). Estradiol (E2) administration also rescued embryo attachment failure in LNG-treated mice (%MAS, 83%). Furthermore, excess P4 treatment before implantation decreased decidual P4 receptor (PGR) expression and induced decidualization defect apart from LIF downregulation. These findings indicate that progestogens cause embryo attachment inhibition through downregulation of uterine LIF expression and compromised decidualization through downregulation of PGR independently of LIF reduction. This study may contribute to a better understanding of contraceptive action of progestogens.
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http://dx.doi.org/10.1210/endocr/bqz005DOI Listing
February 2020

Therapeutic targeting of mitochondrial ROS ameliorates murine model of volume overload cardiomyopathy.

J Pharmacol Sci 2019 Sep 28;141(1):56-63. Epub 2019 Sep 28.

Department of Cardiac Surgery, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-8655, Japan.

Concomitant heart failure is associated with poor clinical outcome in dialysis patients. The arteriovenous shunt, created as vascular access for hemodialysis, increases ventricular volume-overload, predisposing patients to developing cardiac dysfunction. The integral function of mitochondrial respiration is critically important for the heart to cope with hemodynamic overload. The involvement, however, of mitochondrial activity or reactive oxygen species (ROS) in the pathogenesis of ventricular-overload-induced heart failure has not been fully elucidated. We herein report that disorganization of mitochondrial respiration increases mitochondrial ROS production in the volume-overloaded heart, leading to ventricular dysfunction. We adopted the murine arteriovenous fistula (AVF) model, which replicates the cardinal features of volume-overload-induced ventricular dysfunction. Enzymatic assays of cardiac mitochondria revealed that the activities of citrate synthase and NADH-quinone reductase (complex Ⅰ) were preserved in the AVF heart. In contrast, the activity of NADH oxidase supercomplex was significantly compromised, resulting in elevated ROS production. Importantly, the antioxidant N-acetylcysteine prevented the development of ventricular dilatation and cardiac dysfunction, suggesting a pathogenic role for ROS in dialysis-related cardiomyopathy. A cardioprotective effect was also observed in metformin-treated mice, illuminating its potential use in the management of heart failure complicating diabetic patients on dialysis.
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http://dx.doi.org/10.1016/j.jphs.2019.09.005DOI Listing
September 2019

Copper-Catalyzed Cycloisomerization of Cyclopropenylimine for Synthesis of Pyrroles.

J Org Chem 2019 11 8;84(21):14320-14329. Epub 2019 Oct 8.

Kobe Pharmaceutical University, Motoyamakita , Higashinada, Kobe 658-8558 , Japan.

Copper-catalyzed cycloisomerization of 3-iminocyclopropenes for synthesis of pyrroles has been developed. The reaction allows regioselective construction of pyrroles with various substitution patterns, including fully substituted pyrroles. The method was successfully applied to synthesis of steroidal pyrroles as well as a N-fused pyrrole.
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http://dx.doi.org/10.1021/acs.joc.9b02178DOI Listing
November 2019
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