Springer Science and Business Media LLC Acta Medica Okayama 2045-2322 16 1 2026 Nitric oxide induces p53-mediated cell death in human nasal epithelial cells 10055 EN Shizuki Kamiuezono Department of Medicinal Pharmacology, Faculty of Pharmaceutical Sciences, Okayama University Sho Kubota Department of Medicinal Pharmacology, Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama University Tomoki Tsuchida Department of Medicinal Pharmacology, Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama University Nobumasa Takasugi Department of Medicinal Pharmacology, Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama University Takashi Uehara Department of Medicinal Pharmacology, Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama University Nitric oxide (NO) is a key signaling molecule that plays a vital role in maintaining homeostasis of physiological processes such as immune responses and neurotransmission. However, excessive NO production during inflammatory responses to infection can lead to cytotoxicity and tissue damage. The nasal epithelial barrier is a crucial first line of immunological defense against viral infections, and it is likely exposed to excessive NO levels during chronic inflammation. Therefore, clarifying the effects of NO on this barrier is thus critical. In this study, we investigated the biological effects of sustained NO exposure on RPMI2650 human nasal epithelial cells. Post-NO exposure transcriptomic analyses revealed significant upregulation of genes involved in the p53 signaling pathway. RT-qPCR analyses confirmed the temporal upregulation of p53 target genes associated with apoptosis and cell cycle regulation. These gene expression changes downregulated cell proliferation and induced cell death. Our findings suggest that excessive NO exposure induces nasal epithelial cell death via the p53 pathway, which over the long term can result in tissue damage and dysfunction under inflammatory conditions. These results provide new insights into how prolonged NO exposure affects the nasal epithelial cells and may contribute to the progression of chronic infectious diseases. No potential conflict of interest relevant to this article was reported.

Pharmaceutical Society of Japan Acta Medica Okayama 0918-6158 49 1 2026 Exploratory Analysis for Development Predictive Models of Immune Checkpoint Inhibitor-Induced Myocarditis Using a Nationwide Claims Database 66 73 EN Reina Yamamoto Department of Medicinal Pharmacology, Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama University Hirofumi Hamano Department of Pharmacy, Okayama University Hospital Koki Nakagomi Department of Clinical Pharmacy, Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama University Miyu Uchiyama Department of Clinical Pharmacy, Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama University Ayana Michihara Department of Pharmacy, Okayama University Hospital Aya F. Ozaki Department of Clinical Pharmacy Practice, School of Pharmacy & Pharmaceutical Sciences, University of California Pranav M. Patel Division of Cardiology, School of Medicine, University of California Maki Tanioka Medical AI Project, Dentistry and Pharmaceutical Science, Okayama University Graduate School of Medicine Yoshito Zamami Department of Pharmacy, Okayama University Hospital Takashi Uehara Department of Medicinal Pharmacology, Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama University Immune checkpoint inhibitors (ICIs), essential in cancer therapy, can cause severe immune-related adverse events (irAEs), including myocarditis with a high fatality rate. Currently, the pathogenesis, biomarkers, and risk factors of ICI-induced myocarditis (ICIM) are not fully understood. This exploratory study aimed to develop machine learning-based models to predict the onset of ICIM within 3 months of starting ICI therapy, using a large health insurance database. The models were constructed using the Light Gradient Boosting Machine (LightGBM) and Random Forest algorithms, incorporating clinical variables such as comorbidities and prior medication classifications. In this study, a strategy combining undersampling and bagging was used to minimize the impact of highly imbalanced datasets. The Random Forest model demonstrated superior performance compared with the LightGBM model, and the SHapley Additive exPlanations (SHAP) analysis for the Random Forest model revealed that the concurrent use of ICIs was the most important variable for predictions. Although predictive performance remains limited (AUROC ≈ 0.63), this exploratory framework demonstrates the feasibility of developing data-driven risk prediction models for ICIM. Future studies with expanded datasets and integration of laboratory parameters are warranted to improve predictive accuracy and potential clinical applicability. No potential conflict of interest relevant to this article was reported.

American Chemical Society (ACS) Acta Medica Okayama 1554-8929 19 12 2024 Discovery of a Compound That Inhibits IRE1α S-Nitrosylation and Preserves the Endoplasmic Reticulum Stress Response under Nitrosative Stress 2429 2437 EN Haruna Kurogi Department of Medicinal Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Nobumasa Takasugi Department of Medicinal Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Sho Kubota Department of Medicinal Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Ashutosh Kumar Laboratory for Structural Bioinformatics, Center for Biosystems Dynamics Research, RIKEN Takehiro Suzuki Biomolecular Characterization Unit, Technology Platform Division, RIKEN Center for Sustainable Resource Science Naoshi Dohmae Biomolecular Characterization Unit, Technology Platform Division, RIKEN Center for Sustainable Resource Science Daisuke Sawada Department of Fine Organic Synthesis, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Kam Y.J. Zhang Laboratory for Structural Bioinformatics, Center for Biosystems Dynamics Research, RIKEN Takashi Uehara Department of Medicinal Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Inositol-requiring enzyme 1α (IRE1α) is a sensor of endoplasmic reticulum (ER) stress and drives ER stress response pathways. Activated IRE1α exhibits RNase activity and cleaves mRNA encoding X-box binding protein 1, a transcription factor that induces the expression of genes that maintain ER proteostasis for cell survival. Previously, we showed that IRE1α undergoes S-nitrosylation, a post-translational modification induced by nitric oxide (NO), resulting in reduced RNase activity. Therefore, S-nitrosylation of IRE1α compromises the response to ER stress, making cells more vulnerable. We conducted virtual screening and cell-based validation experiments to identify compounds that inhibit the S-nitrosylation of IRE1α by targeting nitrosylated cysteine residues. We ultimately identified a compound (1ACTA) that selectively inhibits the S-nitrosylation of IRE1α and prevents the NO-induced reduction of RNase activity. Furthermore, 1ACTA reduces the rate of NO-induced cell death. Our research identified S-nitrosylation as a novel target for drug development for IRE1α and provides a suitable screening strategy. No potential conflict of interest relevant to this article was reported.

MDPI Acta Medica Okayama 1661-6596 25 21 2024 Epigenetic Regulation of CXC Chemokine Expression by Environmental Electrophiles Through DNA Methyltransferase Inhibition 11592 EN Tomoki Tsuchida Department of Medicinal Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Sho Kubota Department of Medicinal Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Shizuki Kamiuezono Department of Medicinal Pharmacology, Faculty of Pharmaceutical Sciences, Okayama University Nobumasa Takasugi Department of Medicinal Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Akihiro Ito School of Life Sciences, Tokyo University of Pharmacy and Life Sciences Yoshito Kumagai Graduate School of Pharmaceutical Sciences, Kyushu University Takashi Uehara Department of Medicinal Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Ubiquitously distributed environmental electrophiles covalently modify DNA and proteins, potentially leading to adverse health effects. However, the impacts of specific electrophiles on target proteins and their physiological roles remain largely unknown. In the present study, we focused on DNA methylation, which regulates gene expression and physiological responses. A total of 45 environmental electrophiles were screened for inhibitory effects on the activity of DNA methyltransferase 3B (DNMT3B), a key enzyme in DNA methylation, and four compounds were identified. We focused on 1,2-naphthoquinone (1,2-NQ), an air pollutant whose toxicity has been reported previously. Interestingly, we found that 1,2-NQ modified multiple lysine and histidine residues in DNMT3B, one of which was near the active site in DNMT3B. It was found that 1,2-NQ altered gene expression and evoked inflammatory responses in lung adenocarcinoma cell lines. Furthermore, we found that 1,2-NQ upregulated CXCL8 expression through DNA demethylation of the distal enhancer and promoted cancer cell growth. Our study reveals novel mechanisms of epigenetic regulation by environmental electrophiles through the inhibition of DNMT3B activity and suggests their physiological impact. No potential conflict of interest relevant to this article was reported.

Elsevier Acta Medica Okayama 1083-351X 300 3 2024 Methyl vinyl ketone and its analogs covalently modify PI3K and alter physiological functions by inhibiting PI3K signaling 105679 EN Atsushi Morimoto Department of Medicinal Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Nobumasa Takasugi Department of Medicinal Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Yuexuan Pan Department of Medicinal Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Sho Kubota Department of Medicinal Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Naoshi Dohmae Biomolecular Characterization Unit, Technology Platform Division, RIKEN Center for Sustainable Resource Science Yumi Abiko Graduate School of Biomedical Science, Nagasaki University Koji Uchida Laboratory of Food Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo Yoshito Kumagai Graduate School of Pharmaceutical Sciences, Kyushu University Takashi Uehara Department of Medicinal Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Reactive carbonyl species (RCS), which are abundant in the environment and are produced in vivo under stress, covalently bind to nucleophilic residues such as Cys in proteins. Disruption of protein function by RCS exposure is predicted to play a role in the development of various diseases such as cancer and metabolic disorders, but most studies on RCS have been limited to simple cytotoxicity validation, leaving their target proteins and resulting physiological changes unknown. In this study, we focused on methyl vinyl ketone (MVK), which is one of the main RCS found in cigarette smoke and exhaust gas. We found that MVK suppressed PI3K-Akt signaling, which regulates processes involved in cellular homeostasis, including cell proliferation, autophagy, and glucose metabolism. Interestingly, MVK inhibits the interaction between the epidermal growth factor receptor and PI3K. Cys656 in the SH2 domain of the PI3K p85 subunit, which is the covalently binding site of MVK, is important for this interaction. Suppression of PI3K- Akt signaling by MVK reversed epidermal growth factor- induced negative regulation of autophagy and attenuated glucose uptake. Furthermore, we analyzed the effects of the 23 RCS compounds with structures similar to MVK and showed that their analogs also suppressed PI3K-Akt signaling in a manner that correlated with their similarities to MVK. Our study demonstrates the mechanism of MVK and its analogs in suppressing PI3K-Akt signaling and modulating physiological functions, providing a model for future studies analyzing environmental reactive species. No potential conflict of interest relevant to this article was reported.

Springer Science and Business Media LLC Acta Medica Okayama 0340-5761 99 2 2024 Therapeutic potential of 4-phenylbutyric acid against methylmercury-induced neuronal cell death in mice 563 574 EN Ryohei Miki Department of Medicinal Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Ryosuke Nomura Department of Medicinal Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Yuta Iijima Department of Medicinal Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Sho Kubota Department of Medicinal Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Nobumasa Takasugi Department of Medicinal Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Takao Iwawaki Division of Cell Medicine, Department of Life Science, Medical Research Institute, Kanazawa Medical University Masatake Fujimura Department of International Affairs and Research, National Institute for Minamata Disease Takashi Uehara Department of Medicinal Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Methylmercury (MeHg) is an environmental neurotoxin that induces damage to the central nervous system and is the causative agent in Minamata disease. The mechanisms underlying MeHg neurotoxicity remain largely unknown, and there is a need for effective therapeutic agents, such as those that target MeHg-induced endoplasmic reticulum (ER) stress and the unfolded protein response (UPR), which is activated as a defense mechanism. We investigated whether intraperitoneal administration of the chemical chaperone, 4-phenylbutyric acid (4-PBA), at 120 mg/kg/day can alleviate neurotoxicity in the brains of mice administered 50 ppm MeHg in drinking water for 5 weeks. 4-PBA significantly reduced MeHg-induced ER stress, neuronal apoptosis, and neurological symptoms. Furthermore, 4-PBA was effective even when administered 2 weeks after the initiation of exposure to 30 ppm MeHg in drinking water. Our results strongly indicate that ER stress and the UPR are key processes involved in MeHg toxicity, and that 4-PBA is a novel therapeutic candidate for MeHg-induced neurotoxicity. No potential conflict of interest relevant to this article was reported.

Pharmaceutical Society of Japan Acta Medica Okayama 0918-6158 47 6 2024 Epigenetic Regulation of Carbonic Anhydrase 9 Expression by Nitric Oxide in Human Small Airway Epithelial Cells 1119 1122 EN Yuto Moriya Department of Medicinal Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Sho Kubota Department of Medicinal Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Yuta Iijima Department of Medicinal Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Nobumasa Takasugi Department of Medicinal Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Takashi Uehara Department of Medicinal Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University DNA methylation is a crucial epigenetic modification that regulates gene expression and determines cell fate; however, the triggers that alter DNA methylation levels remain unclear. Recently, we showed that S-nitrosylation of DNA methyltransferase (DNMT) induces DNA hypomethylation and alters gene expression. Furthermore, we identified DBIC, a specific inhibitor of S-nitrosylation of DNMT3B, to suppress nitric oxide (NO)-induced gene alterations. However, it remains unclear how NO-induced DNA hypomethylation regulates gene expression and whether this mechanism is maintained in normal cells and triggers disease-related changes. To address these issues, we focused on carbonic anhydrase 9 (CA9), which is upregulated under nitrosative stress in cancer cells. We pharmacologically evaluated its regulatory mechanisms using human small airway epithelial cells (SAECs) and DBIC. We demonstrated that nitrosative stress promotes the recruitment of hypoxia-inducible factor 1 alpha to the CA9 promoter region and epigenetically induces CA9 expression in SAECs. Our results suggest that nitrosative stress is a key epigenetic regulator that may cause diseases by altering normal cell function. No potential conflict of interest relevant to this article was reported.

Japanese Pharmacological Society Acta Medica Okayama 1347-8613 154 3 2024 Attenuation of protein arginine dimethylation via S-nitrosylation of protein arginine methyltransferase 1 209 217 EN Rikako Taniguchi Department of Medicinal Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Yuto Moriya Department of Medicinal Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Naoshi Dohmae Biomolecular Characterization Unit, Technology Platform Division, RIKEN Center for Sustainable Resource Science Takehiro Suzuki Biomolecular Characterization Unit, Technology Platform Division, RIKEN Center for Sustainable Resource Science Kengo Nakahara Department of Medicinal Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Sho Kubota Department of Medicinal Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Nobumasa Takasugi Department of Medicinal Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Takashi Uehara Department of Medicinal Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Upregulation of nitric oxide (NO) production contributes to the pathogenesis of numerous diseases via S-nitro- sylation, a post-translational modification of proteins. This process occurs due to the oxidative reaction between NO and a cysteine thiol group; however, the extent of this reaction remains unknown. S-Nitrosylation of PRMT1, a major asymmetric arginine methyltransferase of histones and numerous RNA metabolic proteins, was induced by NO donor treatment. We found that nitrosative stress leads to S-nitrosylation of cysteine 119, located near the active site, and attenuates the enzymatic activity of PRMT1. Interestingly, RNA sequencing analysis revealed similarities in the changes in expression elicited by NO and PRMT1 inhibitors or knockdown. A comprehensive search for PRMT1 substrates using the proximity-dependent biotin identification method highlighted many known and new substrates, including RNA-metabolizing enzymes. To validate this result, we selected the RNA helicase DDX3 and demonstrated that arginine methylation of DDX3 is induced by PRMT1 and attenuated by NO treatment. Our results suggest the existence of a novel regulatory system associated with transcription and RNA metabolism via protein S-nitrosylation. No potential conflict of interest relevant to this article was reported.

Japanese Society of Toxicology Acta Medica Okayama 0388-1350 49 2 2024 Methylmercury-induced brain neuronal death in CHOP-knockout mice 55 60 EN Yuta Iijima Department of Medicinal Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Ryohei Miki Department of Medicinal Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Masatake Fujimura Department of Basic Medical Science, National Institute for Minamata Disease Seiichi Oyadomari Division of Molecular Biology, Institute of Advanced Medical Sciences, Tokushima University Takashi Uehara Department of Medicinal Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Apoptosis is one of the hallmarks of MeHg-induced neuronal cell death; however, its molecular mechanism remains unclear. We previously reported that MeHg exposure induces neuron-specific ER stress in the mouse brain. Excessive ER stress contributes to apoptosis, and CHOP induction is considered to be one of the major mechanisms. CHOP is also increased by MeHg exposure in the mouse brain, suggesting that it correlates with increased apoptosis. In this study, to clarify whether CHOP mediates MeHg-induced apoptosis, we examined the effect of CHOP deletion on MeHg exposure in CHOP-knockout mice. Our data showed that CHOP deletion had no effect on MeHg exposure-induced weight loss or hindlimb impairment in mice, nor did it increase apoptosis or inhibit neuronal cell loss. Hence, CHOP plays little role in MeHg toxicity, and other apoptotic pathways coupled with ER stress may be involved in MeHg-induced cell death. No potential conflict of interest relevant to this article was reported.

Nature Portfolio Acta Medica Okayama 2041-1723 14 1 2023 Pivotal role for S-nitrosylation of DNA methyltransferase 3B in epigenetic regulation of tumorigenesis 621 EN Kosaku Okuda Department of Medicinal Pharmacology, Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama University Kengo Nakahara Department of Medicinal Pharmacology, Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama University Akihiro Ito Chemical Genomics Research Group, RIKEN Center for Sustainable Resource Science Yuta Iijima Department of Medicinal Pharmacology, Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama University Ryosuke Nomura Department of Medicinal Pharmacology, Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama University Ashutosh Kumar Laboratory for Structural Bioinformatics, Center for Biosystems Dynamics Research, RIKEN Kana Fujikawa Department of Medicinal Pharmacology, Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama University Kazuya Adachi Department of Medicinal Pharmacology, Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama University Yuki Shimada Department of Medicinal Pharmacology, Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama University Satoshi Fujio Department of Medicinal Pharmacology, Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama University Reina Yamamoto Department of Medicinal Pharmacology, Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama University Nobumasa Takasugi Department of Medicinal Pharmacology, Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama University Kunishige Onuma Division of Experimental Pathology, Faculty of Medicine, Tottori University Mitsuhiko Osaki Division of Experimental Pathology, Faculty of Medicine, Tottori University Futoshi Okada Division of Experimental Pathology, Faculty of Medicine, Tottori University Taichi Ukegawa Department of Synthetic and Medicinal Chemistry, Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama University Yasuo Takeuchi Department of Synthetic and Medicinal Chemistry, Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama University Norihisa Yasui Laboratory of Structural Biology, Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama University Atsuko Yamashita Laboratory of Structural Biology, Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama University Hiroyuki Marusawa Department of Gastroenterology and Hepatology, Graduate School of Medicine, Kyoto University Yosuke Matsushita Division of Genome Medicine, Institute of Advanced Medical Sciences, Tokushima University Toyomasa Katagiri Division of Genome Medicine, Institute of Advanced Medical Sciences, Tokushima University Takahiro Shibata Graduate School of Bioagricultural Sciences, Nagoya University Koji Uchida Laboratory of Food Chemistry, Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo Sheng-Yong Niu Broad Institute of MIT and Harvard Nhi B. Lang Neurodegeneration New Medicines Center, and Departments of Molecular Medicine and Neuroscience, The Scripps Research Institute Tomohiro Nakamura Neurodegeneration New Medicines Center, and Departments of Molecular Medicine and Neuroscience, The Scripps Research Institute Kam Y. J. Zhang Laboratory for Structural Bioinformatics, Center for Biosystems Dynamics Research, RIKEN Stuart A. Lipton Neurodegeneration New Medicines Center, and Departments of Molecular Medicine and Neuroscience, The Scripps Research Institute Takashi Uehara Department of Medicinal Pharmacology, Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama University DNA methyltransferases (DNMTs) catalyze methylation at the C5 position of cytosine with S-adenosyl-l-methionine. Methylation regulates gene expression, serving a variety of physiological and pathophysiological roles. The chemical mechanisms regulating DNMT enzymatic activity, however, are not fully elucidated. Here, we show that protein S-nitrosylation of a cysteine residue in DNMT3B attenuates DNMT3B enzymatic activity and consequent aberrant upregulation of gene expression. These genes include Cyclin D2 (Ccnd2), which is required for neoplastic cell proliferation in some tumor types. In cell-based and in vivo cancer models, only DNMT3B enzymatic activity, and not DNMT1 or DNMT3A, affects Ccnd2 expression. Using structure-based virtual screening, we discovered chemical compounds that specifically inhibit S-nitrosylation without directly affecting DNMT3B enzymatic activity. The lead compound, designated DBIC, inhibits S-nitrosylation of DNMT3B at low concentrations (IC50 <= 100nM). Treatment with DBIC prevents nitric oxide (NO)-induced conversion of human colonic adenoma to adenocarcinoma in vitro. Additionally, in vivo treatment with DBIC strongly attenuates tumor development in a mouse model of carcinogenesis triggered by inflammation-induced generation of NO. Our results demonstrate that de novo DNA methylation mediated by DNMT3B is regulated by NO, and DBIC protects against tumor formation by preventing aberrant S-nitrosylation of DNMT3B. No potential conflict of interest relevant to this article was reported.

MDPI Acta Medica Okayama 2073-4409 12 3 2023 The Pursuit of the "Inside" of the Amyloid Hypothesis-Is C99 a Promising Therapeutic Target for Alzheimer's Disease? 454 EN Nobumasa Takasugi Department of Medicinal Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Masato Komai Department of Medicinal Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Nanaka Kaneshiro Division of Biomedical Sciences, School of Medicine, University of California Atsuya Ikeda Department of Medicinal Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Yuji Kamikubo Department of Cellular and Molecular Pharmacology, Juntendo University Graduate School of Medicine Takashi Uehara Department of Medicinal Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Aducanumab, co-developed by Eisai (Japan) and Biogen (U.S.), has received Food and Drug Administration approval for treating Alzheimer's disease (AD). In addition, its successor antibody, lecanemab, has been approved. These antibodies target the aggregated form of the small peptide, amyloid-beta (A beta), which accumulates in the patient brain. The "amyloid hypothesis " based therapy that places the aggregation and toxicity of A beta at the center of the etiology is about to be realized. However, the effects of immunotherapy are still limited, suggesting the need to reconsider this hypothesis. A beta is produced from a type-I transmembrane protein, A beta precursor protein (APP). One of the APP metabolites, the 99-amino acids C-terminal fragment (C99, also called beta CTF), is a direct precursor of A beta and accumulates in the AD patient's brain to demonstrate toxicity independent of A beta. Conventional drug discovery strategies have focused on A beta toxicity on the "outside " of the neuron, but C99 accumulation might explain the toxicity on the "inside " of the neuron, which was overlooked in the hypothesis. Furthermore, the common region of C99 and A beta is a promising target for multifunctional AD drugs. This review aimed to outline the nature, metabolism, and impact of C99 on AD pathogenesis and discuss whether it could be a therapeutic target complementing the amyloid hypothesis. No potential conflict of interest relevant to this article was reported.

MDPI Acta Medica Okayama 1422-0067 23 23 2022 Alterations in UPR Signaling by Methylmercury Trigger Neuronal Cell Death in the Mouse Brain 15412 EN Ryosuke Nomura Department of Medicinal Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Nobumasa Takasugi Department of Medicinal Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Hideki Hiraoka Department of Medicinal Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Yuta Iijima Department of Medicinal Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Takao Iwawaki Division of Cell Medicine, Department of Life Science, Medical Research Institute, Kanazawa Medical University Yoshito Kumagai Environmental Biology Laboratory, Faculty of Medicine, University of Tsukuba Masatake Fujimura Department of Basic Medical Science, National Institute for Minamata Disease Takashi Uehara Department of Medicinal Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Methylmercury (MeHg), an environmental toxicant, induces neuronal cell death and injures specific areas of the brain. MeHg is known to induce oxidative and endoplasmic reticulum (ER) stress. The unfolded protein response (UPR) pathway has a dual nature in that it regulates and protects cells from an overload of improperly folded proteins in the ER, whereas excessively stressed cells are eliminated by apoptosis. Oxidative stress/ER stress induced by methylmercury exposure may tilt the UPR toward apoptosis, but there is little in vivo evidence of a direct link to actual neuronal cell death. Here, by using the ER stress-activated indicator (ERAI) system, we investigated the time course signaling alterations of UPR in vivo in the most affected areas, the somatosensory cortex and striatum. In the ERAI-Venus transgenic mice exposed to MeHg (30 or 50 ppm in drinking water), the ERAI signal, which indicates the activation of the cytoprotective pathway of the UPR, was only transiently enhanced, whereas the apoptotic pathway of the UPR was persistently enhanced. Furthermore, detailed analysis following the time course showed that MeHg-induced apoptosis is strongly associated with alterations in UPR signaling. Our results suggest that UPR modulation could be a therapeutic target for treating neuropathy. No potential conflict of interest relevant to this article was reported.

Cell Press Acta Medica Okayama 2589-0042 25 3 2022 Lipid flippase dysfunction as a therapeutic target for endosomal anomalies in Alzheimer's disease 103869 EN Nanaka Kaneshiro Department of Medicinal Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Masato Komai Department of Medicinal Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Ryosuke Imaoka Department of Medicinal Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Atsuya Ikeda Department of Medicinal Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Yuji Kamikubo Department of Cellular and Molecular Pharmacology, Juntendo University Graduate School of Medicine Takashi Saito Department of Neurocognitive Science, Nagoya City University Graduate School of Medical Sciences Takaomi C. Saido Laboratory for Proteolytic Neuroscience, RIKEN Center for Brain Science Taisuke Tomita Laboratory of Neuropathology and Neuroscience, Graduate School of Pharmaceutical Sciences, The University of Tokyo Tadafumi Hashimoto Department of Neuropathology, Graduate School of Medicine, The University of Tokyo Takeshi Iwatsubo Department of Neuropathology, Graduate School of Medicine, The University of Tokyo Takashi Sakurai Department of Cellular and Molecular Pharmacology, Juntendo University Graduate School of Medicine Takashi Uehara Nobumasa Takasugi Department of Medicinal Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Endosomal anomalies because of vesicular traffic impairment have been indicated as an early pathology of Alzheimer'vertical bar disease (AD). However, the mechanisms and therapeutic targets remain unclear. We previously reported thatbCTF, one of the pathogenic metabolites of APP, interacts with TMEM30A. TMEM30A constitutes a lipid flippase with P4-ATPase and regulates vesicular trafficking through the asymmetric distribution of phospholipids. Therefore, the alteration of lipid flippase activity in AD pathology has got attention. Herein, we showed that the interaction between beta CTF and TMEM30A suppresses the physiological formation and activity of lipid flippase in AD model cells, A7, and App(NLG-F/NLG-F) model mice. Furthermore, the T-RAP peptide derived from the beta CTF binding site of TMEM30A improved endosomal anomalies, which could be a result of the restored lipid flippase activity. Our results provide insights into the mechanisms of vesicular traffic impairment and suggest a therapeutic target for AD. No potential conflict of interest relevant to this article was reported.

Elsevier Acta Medica Okayama 00219258 296 2021 Covalent N-arylation by the pollutant 1,2-naphthoquinone activates the EGF receptor 100524 EN Kengo Nakahara Department of Medicinal Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Kyohei Hamada Department of Medicinal Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Tomoki Tsuchida Department of Medicinal Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Nobumasa Takasugi Department of Medicinal Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Yumi Abiko Environmental Biology Laboratory, Faculty of Medicine, University of Tsukuba Kazuhiko Shien Department of Thoracic, Breast and Endocrinological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Shinichi Toyooka Department of Thoracic, Breast and Endocrinological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Yoshito Kumagai Environmental Biology Laboratory, Faculty of Medicine, University of Tsukuba Takashi Uehara Department of Medicinal Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University The epidermal growth factor receptor (EGFR) is the most intensively investigated receptor tyrosine kinase. Several EGFR mutations and modifications have been shown to lead to abnormal self-activation, which plays a critical role in carcinogenesis. Environmental air pollutants, which are associated with cancer and respiratory diseases, can also activate EGFR. Specifically, the environmental electrophile 1,2-naphthoquinone (1,2-NQ), a component of diesel exhaust particles and particulate matter more generally, has previously been shown to impact EGFR signaling. However, the detailed mechanism of 1,2-NQ function is unknown. Here, we demonstrate that 1,2-NQ is a novel chemical activator of EGFR but not other EGFR family proteins. We found that 1,2-NQ forms a covalent bond, in a reaction referred to as N-arylation, with Lys80, which is in the ligand-binding domain. This modification activates the EGFR–Akt signaling pathway, which inhibits serum deprivation–induced cell death in a human lung adenocarcinoma cell line. Our study reveals a novel mode of EGFR pathway activation and suggests a link between abnormal EGFR activation and environmental pollutant–associated diseases such as cancer. No potential conflict of interest relevant to this article was reported.

Springer Acta Medica Okayama 0340-5761 95 4 2021 Spatiotemporal analysis of the UPR transition induced by methylmercury in the mouse brain 1241 1250 EN Hideki Hiraoka Department of Medicinal Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Ryosuke Nomura Department of Medicinal Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Nobumasa Takasugi Department of Medicinal Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Ryoko Akai Division of Cell Medicine, Department of Life Science, Medical Research Institute, Kanazawa Medical University Takao Iwawaki Division of Cell Medicine, Department of Life Science, Medical Research Institute, Kanazawa Medical University Yoshito Kumagai Environmental Biology Laboratory, Faculty of Medicine, University of Tsukuba Masatake Fujimura Department of Basic Medical Science, National Institute for Minamata Disease Takashi Uehara Department of Medicinal Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Methylmercury (MeHg), an environmental toxicant, induces neuronal cell death and injures a specific area of the brain. MeHg-mediated neurotoxicity is believed to be caused by oxidative stress and endoplasmic reticulum (ER) stress but the mechanism by which those stresses lead to neuronal loss is unclear. Here, by utilizing the ER stress-activated indicator (ERAI) system, we investigated the signaling alterations in the unfolded protein response (UPR) prior to neuronal apoptosis in the mouse brain. In ERAI transgenic mice exposed to MeHg (25 mg/kg, S.C.), the ERAI signal, which indicates activation of the cytoprotective pathway of the UPR, was detected in the brain. Interestingly, detailed ex vivo analysis showed that the ERAI signal was localized predominantly in neurons. Time course analysis of MeHg exposure (30 ppm in drinking water) showed that whereas the ERAI signal was gradually attenuated at the late phase after increasing at the early phase, activation of the apoptotic pathway of the UPR was enhanced in proportion to the exposure time. These results suggest that MeHg induces not only ER stress but also neuronal cell death via a UPR shift. UPR modulation could be a therapeutic target for treating neuropathy caused by electrophiles similar to MeHg. No potential conflict of interest relevant to this article was reported.

Nature Publishing Group Acta Medica Okayama 2045-2322 5 2015 Regulation of the unfolded protein response via S-nitrosylation of sensors of endoplasmic reticulum stress 14812 EN Nakato Ryosuke Ohkubo Yu Konishi Akari Shibata Mari Kaneko Yuki Iwawaki Takao Nakamura Tomohiro Lipton Stuart A. Uehara Takashi Protein S-nitrosylation modulates important cellular processes, including neurotransmission, vasodilation, proliferation, and apoptosis in various cell types. We have previously reported that protein disulfide isomerase (PDI) is S-nitrosylated in brains of patients with sporadic neurodegenerative diseases. This modification inhibits PDI enzymatic activity and consequently leads to the accumulation of unfolded/misfolded proteins in the endoplasmic reticulum (ER) lumen. Here, we describe S-nitrosylation of additional ER pathways that affect the unfolded protein response (UPR) in cell-based models of Parkinson's disease (PD). We demonstrate that nitric oxide (NO) can S-nitrosylate the ER stress sensors IRE1α and PERK. While S-nitrosylation of IRE1α inhibited its ribonuclease activity, S-nitrosylation of PERK activated its kinase activity and downstream phosphorylation/inactivation or eIF2α. Site-directed mutagenesis of IRE1α(Cys931) prevented S-nitrosylation and inhibition of its ribonuclease activity, indicating that Cys931 is the predominant site of S-nitrosylation. Importantly, cells overexpressing mutant IRE1α(C931S) were resistant to NO-induced damage. Our findings show that nitrosative stress leads to dysfunctional ER stress signaling, thus contributing to neuronal cell death. No potential conflict of interest relevant to this article was reported.