There is evidence supporting the notion that excess abdominal fat is predictive of insulin resistance and the presence of related metabolic abnormalities currently referred to as the metabolic syndrome (MetS). Despite the fact that abdominal obesity is a highly prevalent feature of MetS, the mechanisms by which abdominal obesity is causally related to MetS are not fully elucidated. Besides visceral fat accumulation, ectopic lipid deposition, especially in the liver and skeletal muscle, has been implicated in the pathophysiology of diabetes, insulin resistance and obesity-related disorders. In addition, ectopic fat deposition play a critical role in the heart components such as (1) circulatory and locally recruited fat, (2) intra-and extra-myocellular fat, (3) perivascular fat, and (4) pericardial fat. In this review, the contribution of ectopic lipid deposition to global cardiometabolic risk is discussed via possible mechanisms including adipocytokine, insulin resistance and lipotoxicity.
Takashi Tanaka, Thi Dinh Nguyen, Nichakarn Kwankaew, Megumi Sumizono, Reika Shinoda, Hiroshi Ishii, Mika Takarada-Iemata, Tsuyoshi Hattori, Seiichi Oyadomari, Nobuo Kato, Kazutoshi Mori and Osamu Hori : ATF6β Deficiency Elicits Anxiety-like Behavior and Hyperactivity Under Stress Conditions., Neurochemical Research, Vol.48, No.7, 2175-2186, 2023.
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mice. These findings suggest that ATF6β deficiency produces anxiety-like behavior and hyperactivity via a CRH receptor 1-dependent mechanism. ATF6β could play a role in psychiatric conditions in the emotional centers of the brain.
Mitsuaki Sobajima, Masato Miyake, Yoshimasa Hamada, Kazue Tsugawa, Miho Oyadomari, Ryota Inoue, Jun Shirakawa, Hiroshi Arima and Seiichi Oyadomari : The multifaceted role of ATF4 in regulating glucose-stimulated insulin secretion., Biochemical and Biophysical Research Communications, Vol.611, 165-171, 2022.
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Stress-inducible transcription factor ATF4 is essential for survival and identity of β-cell during stress conditions. However, the physiological role of ATF4 in β-cell function is not yet completely understood. To understand the role of ATF4 in glucose-stimulated insulin secretion (GSIS), β-cell-specific Atf4 knockout (βAtf4KO) mice were phenotypically characterized. Insulin secretion and mechanistic analyses were performed using islets from control Atf4f/f and βAtf4KO mice to assess key regulators for triggering and amplifying signals for GSIS. βAtf4KO mice displayed glucose intolerance due to reduced insulin secretion. Moreover, βAtf4KO islets exhibited a decrease in both the insulin content and first-phase insulin secretion. The analysis of βAtf4KO islets showed that ATF4 is required for insulin production and glucose-stimulated ATP and cAMP production. The results demonstrate that ATF4 contributes to the multifaceted regulatory process in GSIS even under stress-free conditions.
Masato Miyake, Mitsuaki Sobajima, Kiyoe Kurahashi, Akira Shigenaga, Masaya Denda, Akira Otaka, Tomohide Saio, Naoki Sakane, Hidetaka Kosako and Seiichi Oyadomari : Identification of an endoplasmic reticulum proteostasis modulator that enhances insulin production in pancreatic β cells., Cell Chemical Biology, Vol.29, No.6, 996-1009.e9, 2022.
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Perturbation of endoplasmic reticulum (ER) proteostasis is associated with impairment of cellular function in diverse diseases, especially the function of pancreatic β cells in type 2 diabetes. Restoration of ER proteostasis by small molecules shows therapeutic promise for type 2 diabetes. Here, using cell-based screening, we report identification of a chemical chaperone-like small molecule, KM04794, that alleviates ER stress. KM04794 prevented protein aggregation and cell death caused by ER stressors and a mutant insulin protein. We also found that this compound increased intracellular and secreted insulin levels in pancreatic β cells. Chemical biology and biochemical approaches revealed that the compound accumulated in the ER and interacted directly with the ER molecular chaperone BiP. Our data show that this corrector of ER proteostasis can enhance insulin storage and pancreatic β cell function.
Masato Miyake, Jun Zhang, Akihiro Yasue, Satoshi Hisanaga, Kazue Tsugawa, Hiroshi Sakaue, Miho Oyadomari, Hiroshi Kiyonari and Seiichi Oyadomari : Integrated stress response regulates GDF15 secretion from adipocytes, preferentially suppresses appetite for a high-fat diet and improves obesity., iScience, Vol.24, No.12, 2021.
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The eIF2α phosphorylation-dependent integrated stress response (ISR) is a signaling pathway that maintains homeostasis in mammalian cells exposed to various stresses. Here, ISR activation in adipocytes improves obesity and diabetes by regulating appetite in a non-cell-autonomous manner. Adipocyte-specific ISR activation using transgenic mice decreases body weight and improves glucose tolerance and obesity induced by a high-fat diet (HFD) via preferential inhibition of HFD intake. The transcriptome analysis of ISR-activated adipose tissue reveals that growth differentiation factor 15 (GDF15) expression is induced by the ISR through the direct regulation of the transcription factors ATF4 and DDIT3. Deficiency in the GDF15 receptor GFRAL abolishes the adipocyte ISR-dependent preferential inhibition of HFD intake and the anti-obesity effects. Pharmacologically, 10(E), 12(Z)-octadecadienoic acid induces ISR-dependent GDF15 expression in adipocytes and decreases the intake of the HFD. Based on our findings the specific activation of the ISR in adipocytes controls the non-cell-autonomous regulation of appetite.
Keisuke Kitakaze, Miho Oyadomari, Jun Zhang, Yoshimasa Hamada, Yasuhiro Takenouchi, Kazuhito Tsuboi, Mai Inagaki, Masanori Tachikawa, Yoshio Fujitani, Yasuo Okamoto and Seiichi Oyadomari : ATF4-mediated transcriptional regulation protects against β-cell loss during endoplasmic reticulum stress in a mouse model., Molecular Metabolism, Vol.54, 2021.
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To examine the role of ATF4 in vivo, ISR enhancer Sephin1 (5 mg/kg body weight, p.o.) was administered daily for 21 days to Akita mice. We also established β-cell-specific Atf4 knockout (βAtf4-KO) mice that were further crossed with Akita mice. These mice were analyzed for characteristics of diabetes, β-cell function, and morphology of the islets. To identify the downstream factors of ATF4 in β-cells, the islets of βAtf4-KO mice were subjected to cDNA microarray analyses. To examine the transcriptional regulation by ATF4, we also performed in situ PCR analysis of pancreatic sections from mice and ChIP-qPCR analysis of CT215 β-cells.
Yu Saitou, Tetsuya Ikemoto, Kazunori Tokuda, Katsuki Miyazaki, Shin-ichiro Yamada, Satoru Imura, Masato Miyake, Yuji Morine, Seiichi Oyadomari and Mitsuo Shimada : Effective three-dimensional culture of hepatocyte-like cells generated from human adipose-derived mesenchymal stem cells., Journal of Hepato-Biliary-Pancreatic Sciences, Vol.28, No.9, 705-715, 2021.
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The aim of this study was to clarify the effectiveness of a new three-dimensional (3D) culture system for hepatocyte-like cells (HLCs) generated from human adipose-derived mesenchymal stem cells (ADSCs). Human ADSCs (2 × 10 ) with or without 0.1 mg/mL human recombinant peptide μ-piece per well were seeded in a 96-well U-bottom plate and then our three-step differentiation protocol was applied for 21 days. At each step, cell morphology and gene expression were investigated. Mature hepatocyte functions were evaluated after HLC differentiation. These parameters were compared between 2D- and 3D-cultured HLCs, and, DNA microarray analysis was also performed. Finally, HLCs were transplanted in to CCl induced acute liver failure model mice. Two-dimensional-cultured HLCs at day 21 did not have a spindle shape and had formed spheroids after day 6, which gradually increased in size for 3D-cultured HLCs. Definitive endoderm, hepatoblast, and hepatocyte genes showed significantly higher expression in the 3D culture group. Three-dimensional-cultured HLCs also had higher albumin expression, CYP3A4 activity, urea synthesis, and ammonium metabolism, and much higher expression of ion transporter, blood coagulation, and cell communication genes. HLC transplantation improved serum liver function, especially in T-Bil levels, and engrafted into immunodeficient mice with HLA class I positive staining. Our new 3D culture protocol is effective to improve hepatocyte functions. Our HLCs might be promising for clinical cell transplantation to treat metabolic disease.
Shojiro Kitajima, Wendi Sun, Leong Kian Lee, Caifeng Jolene Ho, Seiichi Oyadomari, Takashi Okamoto, Hisao Masai, Lorenz Poellinger and Hiroyuki Kato : A KDM6 inhibitor potently induces ATF4 and its target gene expression through HRI activation and by UTX inhibition., Scientific Reports, Vol.11, No.1, 4538, 2021.
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UTX/KDM6A encodes a major histone H3 lysine 27 (H3K27) demethylase, and is frequently mutated in various types of human cancers. Although UTX appears to play a crucial role in oncogenesis, the mechanisms involved are still largely unknown. Here we show that a specific pharmacological inhibitor of H3K27 demethylases, GSK-J4, induces the expression of transcription activating factor 4 (ATF4) protein as well as the ATF4 target genes (e.g. PCK2, CHOP, REDD1, CHAC1 and TRIB3). ATF4 induction by GSK-J4 was due to neither transcriptional nor post-translational regulation. In support of this view, the ATF4 induction was almost exclusively dependent on the heme-regulated eIF2α kinase (HRI) in mouse embryonic fibroblasts (MEFs). Gene expression profiles with UTX disruption by CRISPR-Cas9 editing and the following stable re-expression of UTX showed that UTX specifically suppresses the expression of the ATF4 target genes, suggesting that UTX inhibition is at least partially responsible for the ATF4 induction. Apoptosis induction by GSK-J4 was partially and cell-type specifically correlated with the activation of ATF4-CHOP. These findings highlight that the anti-cancer drug candidate GSK-J4 strongly induces ATF4 and its target genes via HRI activation and raise a possibility that UTX might modulate cancer formation by regulating the HRI-ATF4 axis.
Munenori Habuta, Akihiro Yasue, T Ken-Ichi Suzuki, Hirofumi Fujita, Keita Sato, Hitomi Kono, Ayuko Takayama, Tetsuya Bando, Satoru Miyaishi, Seiichi Oyadomari, Eiji Tanaka and Hideyo Ohuchi : Fgf10-CRISPR mosaic mutants demonstrate the gene dose-related loss of the accessory lobe and decrease in the number of alveolar type 2 epithelial cells in mouse lung., PLoS ONE, Vol.15, No.10, 2020.
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CRISPR/Cas9-mediated gene editing often generates founder generation (F0) mice that exhibit somatic mosaicism in the targeted gene(s). It has been known that Fibroblast growth factor 10 (Fgf10)-null mice exhibit limbless and lungless phenotypes, while intermediate limb phenotypes (variable defective limbs) are observed in the Fgf10-CRISPR F0 mice. However, how the lung phenotype in the Fgf10-mosaic mutants is related to the limb phenotype and genotype has not been investigated. In this study, we examined variable lung phenotypes in the Fgf10-targeted F0 mice to determine if the lung phenotype was correlated with percentage of functional Fgf10 genotypes. Firstly, according to a previous report, Fgf10-CRISPR F0 embryos on embryonic day 16.5 (E16.5) were classified into three types: type I, no limb; type II, limb defect; and type III, normal limbs. Cartilage and bone staining showed that limb truncations were observed in the girdle, (type I), stylopodial, or zeugopodial region (type II). Deep sequencing of the Fgf10-mutant genomes revealed that the mean proportion of codons that encode putative functional FGF10 was 8.3 ± 6.2% in type I, 25.3 ± 2.7% in type II, and 54.3 ± 9.5% in type III (mean ± standard error of the mean) mutants at E16.5. Histological studies showed that almost all lung lobes were absent in type I embryos. The accessory lung lobe was often absent in type II embryos with other lobes dysplastic. All lung lobes formed in type III embryos. The number of terminal tubules was significantly lower in type I and II embryos, but unchanged in type III embryos. To identify alveolar type 2 epithelial (AECII) cells, known to be reduced in the Fgf10-heterozygous mutant, immunostaining using anti-surfactant protein C (SPC) antibody was performed: In the E18.5 lungs, the number of AECII was correlated to the percentage of functional Fgf10 genotypes. These data suggest the Fgf10 gene dose-related loss of the accessory lobe and decrease in the number of alveolar type 2 epithelial cells in mouse lung. Since dysfunction of AECII cells has been implicated in the pathogenesis of parenchymal lung diseases, the Fgf10-CRISPR F0 mouse would present an ideal experimental system to explore it.
Aki Ichihara, Akihiro Yasue, Silvia Naomi Mitsui Akagi, Daishi Arai, Yoshiyuki Minegishi, Seiichi Oyadomari, Issei Imoto and Eiji Tanaka : The C-terminal region including the MH6 domain of Msx1 regulates skeletal development., Biochemical and Biophysical Research Communications, Vol.526, No.1, 62-69, 2020.
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MSX1 is a causative gene for oligodontia in humans. Although conventional Msx1-deficient mice die neonatally, a mutant mouse lacking the C-terminus MH6 domain of MSX1 (Msx1) showed two different phenotypes; newborn homozygotes with cleft palates died neonatally, whereas those with thin palates remained alive and had craniofacial dysplasia and growth retardation compared with wild-type mice, with most mice dying by the age of 4-5 weeks. In a previously reported case of human oligodontia caused by a heterozygous defect of the Msx1 MH6 domain, a small foramen was observed on the occipital bone. The aim of this study was to test the hypothesis that the Msx1 MH6 domain is involved in bone formation in vivo. In Msx1 mice, cranial suture fusion was delayed at embryonic day 18.5, and the anteroposterior cranial diameter was smaller and long bone length was decreased at 3 weeks of age. The femoral epiphysis showed no change in the trabecular number, but decreased bone mass, bone density, and trabecular width in Msx1 mice. In addition, cancellous bone mass was reduced and the cartilage layer in the growth plate was thinner in Msx1 mice. The mRNA expression levels of major osteoblast and chondrocyte differentiation marker genes were decreased in Msx1 mice compared with wild-type mice. These findings suggest that the C-terminal region including the MH6 domain of MSX1 plays important roles not only in tooth development and palatal fusion, but also in postnatal bone formation.
Emi Ikebe, Sahoko Matsuoka, Kenta Tezuka, Madoka Kuramitsu, Kazu Okuma, Makoto Nakashima, Seiichiro Kobayashi, Junya Makiyama, Makoto Yamagishi, Seiichi Oyadomari, Kaoru Uchimaru and Isao Hamaguchi : Activation of PERK-ATF4-CHOP pathway as a novel therapeutic approach for efficient elimination of HTLV-1-infected cells., Blood Advances, Vol.4, No.9, 1845-1858, 2020.
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Patients with adult T-cell leukemia (ATL) exhibit a poor prognosis and overall survival rate when treated with standard chemotherapy, highlighting the continued requirement for the development of novel safe and effective therapies for human T-cell leukemia virus type 1 (HTLV-1)-related diseases. In this study, we demonstrated that MK-2048, a second-generation HIV-1 integrase (IN) inhibitor, potently and selectively kills HTLV-1-infected cells. Differential transcriptome profiling revealed significantly elevated levels of gene expression of the unfolded protein response (UPR) PKR-like ER kinase (PERK) signaling pathway in ATL cell lines following MK-2048 treatment. We also identified a significant downregulation in glucose regulated protein 78 (GRP78), a master regulator of the UPR in the CD4+CADM1+ HTLV-1-infected cell population of primary HTLV-1 carrier peripheral blood mononuclear cells (PBMCs) (n = 9), suggesting that HTLV-1-infected cells are hypersensitive to endoplasmic reticulum (ER) stress-mediated apoptosis. MK-2048 efficiently reduced proviral loads in primary HTLV-1 carrier PBMCs (n = 4), but had no effect on the total numbers of these cells, indicating that MK-2048 does not affect the proliferation of HTLV-1-uninfected PBMCs. MK-2048 specifically activated the ER stress-related proapoptotic gene, DNA damage-inducible transcript 3 protein (DDIT3), also known as C/EBP homologous protein (CHOP), in HTLV-1-infected but not uninfected cells of HTLV-1-carrier PBMCs. Our findings demonstrated that MK-2048 selectively induces HTLV-1-infected cell apoptosis via the activation of the UPR. This novel regulatory mechanism of the HIV IN inhibitor MK-2048 in HTLV-1-infected cells provides a promising prophylactic and therapeutic target for HTLV-1-related diseases including ATL.
The integrated stress response (ISR) is one of the most important cytoprotective mechanisms and is integrated by phosphorylation of the α subunit of eukaryotic translation initiation factor 2 (eIF2α). Four eIF2α kinases, heme-regulated inhibitor (HRI), double-stranded RNA-dependent protein kinase (PKR), PKR-like endoplasmic reticulum kinase (PERK), and general control nonderepressible 2 (GCN2), are activated in response to several stress conditions. We previously reported that nanosecond pulsed electric fields (nsPEFs) are a potential therapeutic tool for ISR activation. In this study, we examined which eIF2α kinase is activated by nsPEF treatment. To assess the responsible eIF2α kinase, we used previously established eIF2α kinase quadruple knockout (4KO) and single eIF2α kinase-rescued 4KO mouse embryonic fibroblast (MEF) cells. nsPEFs 70 ns in duration with 30 kV/cm electric fields caused eIF2α phosphorylation in wild-type (WT) MEF cells. On the other hand, nsPEF-induced eIF2α phosphorylation was completely abolished in 4KO MEF cells and was recovered by HRI overexpression. CM-H2DCFDA staining showed that nsPEFs generated reactive oxygen species (ROS), which activated HRI. nsPEF-induced eIF2α phosphorylation was blocked by treatment with the ROS scavenger N-acetyl-L-cysteine (NAC). Our results indicate that the eIF2α kinase HRI is responsible for nsPEF-induced ISR activation and is activated by nsPEF-generated ROS.
Hironori Kato, Kohki Okabe, Masato Miyake, Kazuki Hattori, Tomohiro Fukaya, Kousuke Tanimoto, Shi Beini, Mariko Mizuguchi, Satoru Torii, Satoko Arakawa, Masaya Ono, Yusuke Saito, Takashi Sugiyama, Takashi Funatsu, Katsuaki Sato, Shigeomi Shimizu, Seiichi Oyadomari, Hidenori Ichijo, Hisae Kadowaki and Hideki Nishitoh : ER-resident sensor PERK is essential for mitochondrial thermogenesis in brown adipose tissue., Life Science Alliance, Vol.3, No.3, e201900576, 2020.
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Mitochondria play a central role in the function of brown adipocytes (BAs). Although mitochondrial biogenesis, which is indispensable for thermogenesis, is regulated by coordination between nuclear DNA transcription and mitochondrial DNA transcription, the molecular mechanisms of mitochondrial development during BA differentiation are largely unknown. Here, we show the importance of the ER-resident sensor PKR-like ER kinase (PERK) in the mitochondrial thermogenesis of brown adipose tissue. During BA differentiation, PERK is physiologically phosphorylated independently of the ER stress. This PERK phosphorylation induces transcriptional activation by GA-binding protein transcription factor α subunit (GABPα), which is required for mitochondrial inner membrane protein biogenesis, and this novel role of PERK is involved in maintaining the body temperatures of mice during cold exposure. Our findings demonstrate that mitochondrial development regulated by the PERK-GABPα axis is indispensable for thermogenesis in brown adipose tissue.
M Alexandra Stevens, Michael Xiang, N Lisa Heppler, Isidora Tošić, Kevin Jiang, O Jaime Munoz, S Amos Gaikwad, M Terzah Horton, Xin Long, Padmini Narayanan, L Elizabeth Seashore, C Maci Terrell, Raushan Rashid, J Michael Krueger, E Alicia Mangubat-Medina, T Zachary Ball, Pavel Sumazin, R Sarah Walker, Yoshimasa Hamada, Seiichi Oyadomari, S Michele Redell and A David Frank : Atovaquone is active against AML by upregulating the integrated stress pathway and suppressing oxidative phosphorylation., Blood Advances, Vol.3, No.24, 4215-4227, 2019.
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Atovaquone, a US Food and Drug Administration-approved antiparasitic drug previously shown to reduce interleukin-6/STAT3 signaling in myeloma cells, is well tolerated, and plasma concentrations of 40 to 80 µM have been achieved with pediatric and adult dosing. We conducted preclinical testing of atovaquone with acute myeloid leukemia (AML) cell lines and pediatric patient samples. Atovaquone induced apoptosis with an EC50 <30 µM for most AML lines and primary pediatric AML specimens. In NSG mice xenografted with luciferase-expressing THP-1 cells and in those receiving a patient-derived xenograft, atovaquone-treated mice demonstrated decreased disease burden and prolonged survival. To gain a better understanding of the mechanism of atovaquone, we performed an integrated analysis of gene expression changes occurring in cancer cell lines after atovaquone exposure. Atovaquone promoted phosphorylation of eIF2α, a key component of the integrated stress response and master regulator of protein translation. Increased levels of phosphorylated eIF2α led to greater abundance of the transcription factor ATF4 and its target genes, including proapoptotic CHOP and CHAC1. Furthermore, atovaquone upregulated REDD1, an ATF4 target gene and negative regulator of the mechanistic target of rapamycin (mTOR), and caused REDD1-mediated inhibition of mTOR activity with similar efficacy as rapamycin. Additionally, atovaquone suppressed the oxygen consumption rate of AML cells, which has specific implications for chemotherapy-resistant AML blasts that rely on oxidative phosphorylation for survival. Our results provide insight into the complex biological effects of atovaquone, highlighting its potential as an anticancer therapy with novel and diverse mechanisms of action, and support further clinical evaluation of atovaquone for pediatric and adult AML.
Keisuke Kitakaze, Shusuke Taniuchi, Eri Kawano, Yoshimasa Hamada, Masato Miyake, Miho Oyadomari, Hirotatsu Kojima, Hidetaka Kosako, Tomoko Kuribara, Suguru Yoshida, Takamitsu Hosoya and Seiichi Oyadomari : Cell-based HTS identifies a chemical chaperone for preventing ER protein aggregation and proteotoxicity., eLife, Vol.8, e43302, 2019.
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]benzothiazole derivatives (IBTs) as chemical chaperones in a cell-based high-throughput screen. Biochemical and chemical biology approaches revealed that IBT21 directly binds to unfolded or misfolded proteins and inhibits protein aggregation. Finally, IBT21 prevented cell death caused by chemically induced ER stress and by a proteotoxin, an aggression-prone prion protein. Taken together, our data show the promise of IBTs as potent chemical chaperones that can ameliorate diseases resulting from protein aggregation under ER stress.
A Denis Mogilenko, T Joel Haas, Laurent L'homme, Sébastien Fleury, Sandrine Quemener, Matthieu Levavasseur, Coralie Becquart, Julien Wartelle, Alexandra Bogomolova, Laurent Pineau, Olivier Molendi-Coste, Steve Lancel, Hélène Dehondt, Celine Gheeraert, Aurelie Melchior, Cédric Dewas, Artemii Nikitin, Samuel Pic, Nabil Rabhi, Jean-Sébastien Annicotte, Seiichi Oyadomari, Talia Velasco-Hernandez, Jörg Cammenga, Marc Foretz, Benoit Viollet, Milica Vukovic, Arnaud Villacreces, Kamil Kranc, Peter Carmeliet, Guillemette Marot, Alexis Boulter, Simon Tavernier, Luciana Berod, P Maria Longhi, Christophe Paget, Sophie Janssens, Delphine Staumont-Sallé, Ezra Aksoy, Bart Staels and David Dombrowicz : Metabolic and Innate Immune Cues Merge into a Specific Inflammatory Response via the UPR., Cell, Vol.177, No.5, 1201-1216.e19, 2019.
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Innate immune responses are intricately linked with intracellular metabolism of myeloid cells. Toll-like receptor (TLR) stimulation shifts intracellular metabolism toward glycolysis, while anti-inflammatory signals depend on enhanced mitochondrial respiration. How exogenous metabolic signals affect the immune response is unknown. We demonstrate that TLR-dependent responses of dendritic cells (DCs) are exacerbated by a high-fatty-acid (FA) metabolic environment. FAs suppress the TLR-induced hexokinase activity and perturb tricarboxylic acid cycle metabolism. These metabolic changes enhance mitochondrial reactive oxygen species (mtROS) production and, in turn, the unfolded protein response (UPR), leading to a distinct transcriptomic signature with IL-23 as hallmark. Interestingly, chemical or genetic suppression of glycolysis was sufficient to induce this specific immune response. Conversely, reducing mtROS production or DC-specific deficiency in XBP1 attenuated IL-23 expression and skin inflammation in an IL-23-dependent model of psoriasis. Thus, fine-tuning of innate immunity depends on optimization of metabolic demands and minimization of mtROS-induced UPR.
Junsei Mimura, Atsushi Inose-Maruyama, Shusuke Taniuchi, Kunio Kosaka, Hidemi Yoshida, Hiromi Yamazaki, Shuya Kasai, Nobuhiko Harada, J Randal Kaufman, Seiichi Oyadomari and Ken Itoh : Concomitant Nrf2- and ATF4-activation by Carnosic Acid Cooperatively Induces Expression of Cytoprotective Genes., International Journal of Molecular Sciences, Vol.20, No.7, 1706, 2019.
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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.
Osamu Hashimoto, Masayuki Funaba, Kazunari Sekiyama, Satoru Doi, Daichi Shindo, Ryo Satoh, Hiroshi Itoi, Hiroaki Oiwa, Masahiro Morita, Chisato Suzuki, Makoto Sugiyama, Norio Yamakawa, Hitomi Takada, Shigenobu Matsumura, Kazuo Inoue, Seiichi Oyadomari, Hiromu Sugino and Akira Kurisaki : Activin E Controls Energy Homeostasis in Both Brown and White Adipose Tissues as a Hepatokine., Cell Reports, Vol.25, No.5, 1193-1203, 2018.
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Brown adipocyte activation or beige adipocyte emergence in white adipose tissue (WAT) increases energy expenditure, leading to a reduction in body fat mass and improved glucose metabolism. We found that activin E functions as a hepatokine that enhances thermogenesis in response to cold exposure through beige adipocyte emergence in inguinal WAT (ingWAT). Hepatic activin E overexpression activated thermogenesis through Ucp1 upregulation in ingWAT and other adipose tissues including interscapular brown adipose tissue and mesenteric WAT. Hepatic activin E-transgenic mice exhibited improved insulin sensitivity. Inhibin βE gene silencing inhibited cold-induced Ucp1 induction in ingWAT. Furthermore, in vitro experiments suggested that activin E directly stimulated expression of Ucp1 and Fgf21, which was mediated by transforming growth factor-β or activin type I receptors. We uncovered a function of activin E to stimulate energy expenditure through brown and beige adipocyte activation, suggesting a possible preventive or therapeutic target for obesity.
Satoshi Hisanaga, Masato Miyake, Shusuke Taniuchi, Miho Oyadomari, Masatoshi Morimoto, Ryosuke Sato, Jun Hirose, Hiroshi Mizuta and Seiichi Oyadomari : PERK-mediated translational control is required for collagen secretion in chondrocytes., Scientific Reports, Vol.8, No.1, 773, 2018.
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mice displayed reduced collagen in articular cartilage but no differences in chondrocyte proliferation or apoptosis compared to the findings in wild-type mice. PERK inhibition increases misfolded protein levels in the ER, which largely hinder ER-to-Golgi transport. These results suggest that the translational control mediated by PERK is a critical determinant of ECM secretion in chondrocytes.
Carina Fischer, Takahiro Seki, Sharon Lim, Masaki Nakamura, Patrik Andersson, Yunlong Yang, Jennifer Honek, Yangang Wang, Yanyan Gao, Fang Chen, J Nilesh Samani, Jun Zhang, Masato Miyake, Seiichi Oyadomari, Akihiro Yasue, Xuri Li, Yun Zhang, Yizhi Liu and Yihai Cao : A miR-327-FGF10-FGFR2-mediated autocrine signaling mechanism controls white fat browning., Nature Communications, Vol.8, No.1, 2079, 2017.
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Understanding the molecular mechanisms regulating beige adipocyte formation may lead to the development of new therapies to combat obesity. Here, we report a miRNA-based autocrine regulatory pathway that controls differentiation of preadipocytes into beige adipocytes. We identify miR-327 as one of the most downregulated miRNAs targeting growth factors in the stromal-vascular fraction (SVF) under conditions that promote white adipose tissue (WAT) browning in mice. Gain- and loss-of-function experiments reveal that miR-327 targets FGF10 to prevent beige adipocyte differentiation. Pharmacological and physiological -adrenergic stimulation upregulates FGF10 levels and promotes preadipocyte differentiation into beige adipocytes. In vivo local delivery of miR-327 to WATs significantly compromises the beige phenotype and thermogenesis. Contrarily, systemic inhibition of miR-327 in mice induces browning and increases whole-body metabolic rate under thermoneutral conditions. Our data provide mechanistic insight into an autocrine regulatory signaling loop that regulates beige adipocyte formation and suggests that the miR-327-FGF10-FGFR2 signaling axis may be a therapeutic targets for treatment of obesity and metabolic diseases.
As a novel model of muscle wasting, HSA-Fv2E-PERK Tg mice provide a convenient tool for studying the pathogenesis of muscle loss and for assessing putative therapeutics.
Kiyoe Kurahashi, Seika Inoue, Sumiko Yoshida, Yasumasa Ikeda, Kana Morimoto, Ryoko Uemoto, Kazue Ishikawa, Takeshi Kondo, Tomoyuki Yuasa, Itsuro Endo, Masato Miyake, Seiichi Oyadomari, Toshio Matsumoto, Masahiro Abe, Hiroshi Sakaue and Ken-ichi Aihara : The Role of Heparin Cofactor in the Regulation of Insulin Sensitivity and Maintenance of Glucose Homeostasis in Humans and Mice., Journal of Atherosclerosis and Thrombosis, 2017.
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The present studies provide evidence to support the idea that HC plays an important role in the maintenance of glucose homeostasis by regulating insulin sensitivity in both humans and mice. Stimulators of HC production may serve as novel therapeutic tools for the treatment of type 2 diabetes.
Akihiro Yasue, Hitomi Kono, Munenori Habuta, Tetsuya Bando, Keita Sato, Junji Inoue, Seiichi Oyadomari, Sumihare Noji, Eiji Tanaka and Hideyo Ohuchi : Relationship between somatic mosaicism of Pax6 mutation and variable developmental eye abnormalities-an analysis of CRISPR genome-edited mouse embryos., Scientific Reports, Vol.7, No.1, 2017.
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The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein (Cas) system is a rapid gene-targeting technology that does not require embryonic stem cells. To demonstrate dosage effects of the Pax6 gene on eye formation, we generated Pax6-deficient mice with the CRISPR/Cas system. Eyes of founder embryos at embryonic day (E) 16.5 were examined and categorized according to macroscopic phenotype as class 1 (small eye with distinct pigmentation), class 2 (pigmentation without eye globes), or class 3 (no pigmentation and no eyes). Histologically, class 1 eyes were abnormally small in size with lens still attached to the cornea at E16.5. Class 2 eyes had no lens and distorted convoluted retinas. Class 3 eyes had only rudimentary optic vesicle-like tissues or histological anophthalmia. Genotyping of neck tissue cells from the founder embryos revealed somatic mosaicism and allelic complexity for Pax6. Relationships between eye phenotype and genotype were developed. The present results demonstrated that development of the lens from the surface ectoderm requires a higher gene dose of Pax6 than development of the retina from the optic vesicle. We further anticipate that mice with somatic mosaicism in a targeted gene generated by CRISPR/Cas-mediated genome editing will give some insights for understanding the complexity in human congenital diseases that occur in mosaic form.
Silvia Naomi Mitsui Akagi, Akihiro Yasue, Kiyoshi Masuda, Takuya Naruto, Yoshiyuki Minegishi, Seiichi Oyadomari, Sumihare Noji, Issei Imoto and Eiji Tanaka : Novel human mutation and CRISPR/Cas genome-edited mice reveal the importance of C-terminal domain of MSX1 in tooth and palate development., Scientific Reports, Vol.6, 2016.
(要約)
Several mutations, located mainly in the MSX1 homeodomain, have been identified in non-syndromic tooth agenesis predominantly affecting premolars and third molars. We identified a novel frameshift mutation of the highly conserved C-terminal domain of MSX1, known as Msx homology domain 6 (MH6), in a Japanese family with non-syndromic tooth agenesis. To investigate the importance of MH6 in tooth development, Msx1 was targeted in mice with CRISPR/Cas system. Although heterozygous MH6 disruption did not alter craniofacial development, homozygous mice exhibited agenesis of lower incisors with or without cleft palate at E16.5. In addition, agenesis of the upper third molars and the lower second and third molars were observed in 4-week-old mutant mice. Although the upper second molars were present, they were abnormally small. These results suggest that the C-terminal domain of MSX1 is important for tooth and palate development, and demonstrate that that CRISPR/Cas system can be used as a tool to assess causality of human disorders in vivo and to study the importance of conserved domains in genes.
Shusuke Taniuchi, Masato Miyake, Kazue Tsugawa, Miho Oyadomari and Seiichi Oyadomari : Integrated stress response of vertebrates is regulated by four eIF2α kinases., Scientific Reports, Vol.6, 32886, 2016.
(要約)
The integrated stress response (ISR) is a cytoprotective pathway initiated upon phosphorylation of the eukaryotic translation initiation factor 2 (eIF2α) residue designated serine-51, which is critical for translational control in response to various stress conditions. Four eIF2α kinases, namely heme-regulated inhibitor (HRI), protein kinase R (PKR), PKR-like endoplasmic reticulum kinase, (PERK) and general control non-depressible 2 (GCN2), have been identified thus far, and they are known to be activated by heme depletion, viral infection, endoplasmic reticulum stress, and amino acid starvation, respectively. Because eIF2α is phosphorylated under various stress conditions, the existence of an additional eIF2α kinase has been suggested. To validate the existence of the unidentified eIF2α kinase, we constructed an eIF2α kinase quadruple knockout cells (4KO cells) in which the four known eIF2α kinase genes were deleted using the CRISPR/Cas9-mediated genome editing. Phosphorylation of eIF2α was completely abolished in the 4KO cells by various stress stimulations. Our data suggests that the four known eIF2α kinases are sufficient for ISR and that there are no additional eIF2α kinases in vertebrates.
Wenjun Lu, Daisuke Hagiwara, Yoshiaki Morishita, Masayoshi Tochiya, Yoshinori Azuma, Hidetaka Suga, Motomitsu Goto, Ryoichi Banno, Yoshihisa Sugimura, Seiichi Oyadomari, Kazutoshi Mori and Hiroshi Arima : Unfolded protein response in hypothalamic cultures of wild-type and ATF6-knockout mice., Neuroscience Letters, Vol.612, 199-203, 2015.
(要約)
Recent studies suggest that endoplasmic reticulum (ER) stress in the hypothalamus could affect systemic homeostatic regulation in areas such as energy and water balance. Activating transcription factor 6 (ATF6) is an ER stress transducer which increases the expression of ER chaperones and ER-associated degradation (ERAD) components under ER stress. In the present study, we examined the regulation of the unfolding protein response (UPR) in mouse hypothalamic cultures of wild-type (WT) and ATF6(-/-) mice. Thapsigargin (TG), an ER stressor, significantly increased the mRNA expression of immunoglobulin heavy chain binding protein (BiP), spliced X-box binding protein 1 (XBP1), activating transcription factor 4 (ATF4), C/EBP homologous protein (CHOP), and ERAD components, in hypothalamic cultures of WT mice with the same threshold (0.1M) and similar time courses. On the other hand, TG-induced upregulation of BiP and CHOP as well as most ERAD-related genes, but not spliced XBP1 or ATF4, was attenuated in ATF6(-/-) mice compared with WT mice. Our data suggest that all the UPR arms are activated similarly in the mouse hypothalamus under ER stress conditions, where ATF6 regulates the expression of ER chaperones, CHOP, and ERAD components.
Masato Miyake, Akitoshi Nomura, Atsushi Ogura, Kenji Takehana, Yoshihiro Kitahara, Kazuna Takahara, Kazue Tsugawa, Chinobu Miyamoto, Naoko Miura, Ryosuke Sato, Kiyoe Kurahashi, P Heather Harding, Miho Oyadomari, David Ron and Seiichi Oyadomari : Skeletal muscle-specific eukaryotic translation initiation factor 2 phosphorylation controls amino acid metabolism and fibroblast growth factor 21-mediated non-cell-autonomous energy metabolism., The FASEB journal, Vol.30, No.2, 798-812, 2015.
(要約)
The eukaryotic translation initiation factor 2 (eIF2) phosphorylation-dependent integrated stress response (ISR), a component of the unfolded protein response, has long been known to regulate intermediary metabolism, but the details are poorly worked out. We report that profiling of mRNAs of transgenic mice harboring a ligand-activated skeletal muscle-specific derivative of the eIF2 protein kinase R-like ER kinase revealed the expected up-regulation of genes involved in amino acid biosynthesis and transport but also uncovered the induced expression and secretion of a myokine, fibroblast growth factor 21 (FGF21), that stimulates energy consumption and prevents obesity. The link between the ISR and FGF21 expression was further reinforced by the identification of a small-molecule ISR activator that promoted Fgf21 expression in cell-based screens and by implication of the ISR-inducible activating transcription factor 4 in the process. Our findings establish that eIF2 phosphorylation regulates not only cell-autonomous proteostasis and amino acid metabolism, but also affects non-cell-autonomous metabolic regulation by induced expression of a potent myokine.-Miyake, M., Nomura, A., Ogura, A., Takehana, K., Kitahara, Y., Takahara, K., Tsugawa, K., Miyamoto, C., Miura, N., Sato, R., Kurahashi, K., Harding, H. P., Oyadomari, M., Ron, D., Oyadomari, S. Skeletal muscle-specific eukaryotic translation initiation factor 2 phosphorylation controls amino acid metabolism and fibroblast growth factor 21-mediated non-cell-autonomous energy metabolism.
Chisayo Kozuka, Sumito Sunagawa, Rei Ueda, Moritake Higa, Yuzuru Ohshiro, Hideaki Tanaka, Chigusa Shimizu-Okabe, Chitoshi Takayama, Masayuki Matsushita, Masato Tsutsui, Shogo Ishiuchi, Masanori Nakata, Toshihiko Yada, Jun-Ichi Miyazaki, Seiichi Oyadomari, Michio Shimabukuro and Hiroaki Masuzaki : A novel insulinotropic mechanism of whole grain-derived -oryzanol via the suppression of local dopamine D2 receptor signalling in mouse islet., British Journal of Pharmacology, 2015.
(要約)
-Oryzanol exhibited unique anti-diabetic properties. The unexpected effects of -oryzanol on D2 receptor signalling in islets may provide a novel; natural food-based, approach to anti-diabetic therapy.
Chisayo Kozuka, Sumito Sunagawa, Rei Ueda, Moritake Higa, Hideaki Tanaka, Chigusa Shimizu-Okabe, Shogo Ishiuchi, Chitoshi Takayama, Masayuki Matsushita, Masato Tsutsui, Masato Miyazaki, Seiichi Oyadomari, Michio Shimabukuro and Hiroaki Masuzaki : Gamma-oryzanol protects pancreatic -cells against endoplasmic reticulum stress in male mice., Endocrinology, Vol.156, No.4, 1242-1250, 2015.
(要約)
Endoplasmic reticulum (ER) stress is profoundly involved in dysfunction of β-cells under high-fat diet and hyperglycemia. Our recent study in mice showed that γ-oryzanol, a unique component of brown rice, acts as a chemical chaperone in the hypothalamus and improves feeding behavior and diet-induced dysmetabolism. However, the entire mechanism whereby γ-oryzanol improves glucose metabolism throughout the body still remains unclear. In this context, we tested whether γ-oryzanol reduces ER stress and improves function and survival of pancreatic β-cells using murine β-cell line MIN6. In MIN6 cells with augmented ER stress by tunicamycin, γ-oryzanol decreased exaggerated expression of ER stress-related genes and phosphorylation of eukaryotic initiation factor-2α, resulting in restoration of glucose-stimulated insulin secretion and prevention of apoptosis. In islets from high-fat diet-fed diabetic mice, oral administration of γ-oryzanol improved glucose-stimulated insulin secretion on following reduction of exaggerated ER stress and apoptosis. Furthermore, we examined the impact of γ-oryzanol on low-dose streptozotocin-induced diabetic mice, where exaggerated ER stress and resultant apoptosis in β-cells were observed. Also in this model, γ-oryzanol attenuated mRNA level of genes involved in ER stress and apoptotic signaling in islets, leading to amelioration of glucose dysmetabolism. Taken together, our findings demonstrate that γ-oryzanol directly ameliorates ER stress-induced β-cell dysfunction and subsequent apoptosis, highlighting usefulness of γ-oryzanol for the treatment of diabetes mellitus.
Y Inaba, T Furutani, K Kimura, H Watanabe, S Haga, Y Kido, M Matsumoto, Y Yamamoto, K Harada, S Kaneko, Seiichi Oyadomari, M Ozaki, M Kasuga and H Inoue : Growth arrest and DNA damage-inducible 34 regulates liver regeneration in hepatic steatosis in mice., Hepatology, Vol.61, No.4, 1343-1356, 2015.
(要約)
The liver has robust regenerative potential in response to damage, but hepatic steatosis (HS) weakens this potential. We found that the enhanced integrated stress response (ISR) mediated by phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF2α) impairs regeneration in HS and that growth arrest and DNA damage-inducible 34 (Gadd34)-dependent suppression of ISR plays a crucial role in fatty liver regeneration. Although mice fed a high-fat diet for 2 weeks developed moderate fatty liver with no increase in eIF2α phosphorylation before 70% hepatectomy, they showed impaired liver regeneration as a result of reduced proliferation and increased death of hepatocytes with increased phosphorylation of eIF2α and ISR. An increased ISR through Gadd34 knockdown induced C/EBP homologous protein (CHOP)-dependent apoptosis and receptor-interacting protein kinase 3-dependent necrosis, resulting in increased hepatocyte death during fatty liver regeneration. Furthermore, Gadd34 knockdown and increased phosphorylation of eIF2α decreased cyclin D1 protein and reduced hepatocyte proliferation. In contrast, enhancement of Gadd34 suppressed phosphorylation of eIF2α and reduced CHOP expression and hepatocyte apoptosis without affecting hepatocyte proliferation, clearly improving fatty liver regeneration. In more severe fatty liver of leptin receptor-deficient db/db mice, forced expression of hepatic Gadd34 also promoted hepatic regeneration after hepatectomy. Gadd34-mediated regulation of ISR acts as a physiological defense mechanism against impaired liver regeneration resulting from steatosis and is thus a possible therapeutic target for impaired regeneration in HS.
(キーワード)
Animals / Fatty Liver / Liver Regeneration / Male / Mice / Mice, Inbred C57BL / Protein Phosphatase 1
Yoshinori Azuma, Daisuke Hagiwara, Wenjun Lu, Yoshiaki Morishita, Hidetaka Suga, Motomitsu Goto, Ryoichi Banno, Yoshihisa Sugimura, Seiichi Oyadomari, Kazutoshi Mori, Akira Shiota, Naoya Asai, Masahide Takahashi, Yutaka Oiso and Hiroshi Arima : Activating transcription factor 6 is required for the vasopressin neuron system to maintain water balance under dehydration in male mice., Endocrinology, Vol.155, No.12, 4905-4914, 2014.
(要約)
Activating transcription factor 6 (ATF6) is a sensor of endoplasmic reticulum (ER) stress and increases the expression of ER chaperones and molecules related to the ER-associated degradation of unfolded/misfolded proteins. In this study, we used ATF6 knockout (ATF6(-/-)) mice to clarify the role of ATF6 in the arginine vasopressin (AVP) neuron system. Although urine volumes were not different between ATF6(-/-) and wild-type (ATF6(+/+)) mice with access to water ad libitum, they were increased in ATF6(-/-) mice compared with those in ATF6(+/+) mice under intermittent water deprivation (WD) and accompanied by less urine AVP in ATF6(-/-) mice. The mRNA expression of immunoglobulin heavy chain binding protein, an ER chaperone, was significantly increased in the supraoptic nucleus in ATF6(+/+) but not ATF6(-/-) mice after WD. Electron microscopic analyses demonstrated that the ER lumen of AVP neurons was more dilated in ATF6(-/-) mice than in ATF6(+/+) mice after WD. ATF6(-/-) mice that were mated with mice possessing a mutation causing familial neurohypophysial diabetes insipidus (FNDI), which is characterized by progressive polyuria and AVP neuronal loss due to the accumulation of mutant AVP precursor in the ER, manifested increased urine volume under intermittent WD. The aggregate formation in the ER of AVP neurons was further impaired in FNDI/ATF6(-/-) mice compared with that in FNDI mice, and AVP neuronal loss was accelerated in FNDI/ATF6(-/-) mice under WD. These data suggest that ATF6 is required for the AVP neuron system to maintain water balance under dehydration.
We investigated long-term effects of low carbohydrate diets on wild type mice, streptozotocin-injected and KKAy obese diabetic mice. These mice were pair-fed three different types of diets, standard chow (SC, C∶P∶F = 63∶15∶22), a low carbohydrate (LC, C∶P∶F = 38∶25∶37) diet and a severely carbohydrate restricted (SR, C∶P∶F = 18∶45∶37) diet for 16 weeks. Despite comparable body weights and serum lipid profiles, wild type and diabetic mice fed the low carbohydrate diets exhibited lower insulin sensitivity and this reduction was dependent on the amount of carbohydrate in the diet. When serum fatty acid compositions were investigated, monounsaturation capacity, i.e. C16:1/C16:0 and C18:1/C18:0, was impaired in all murine models fed the low carbohydrate diets, consistent with the decreased expression of hepatic stearoyl-CoA desaturase-1 (SCD1). Interestingly, both the hepatic expressions and serum levels of fibroblast growth factor 21 (FGF21), which might be related to longevity, were markedly decreased in both wild type and KKAy mice fed the SR diet. Taking into consideration that fat compositions did not differ between the LC and SR diets, we conclude that low carbohydrate diets have deleterious metabolic effects in both wild type and diabetic mice, which may explain the association between diets relatively low in carbohydrate and the elevated risk of cardiovascular events observed in clinical studies.
Akihiro Yasue, Silvia Naomi Mitsui Akagi, Takahito Watanabe, Tetsushi Sakuma, Seiichi Oyadomari, Takashi Yamamoto, Sumihare Noji, Taro Mito and Eiji Tanaka : Highly efficient targeted mutagenesis in one-cell mouse embryos mediated by the TALEN and CRISPR/Cas systems., Scientific Reports, Vol.4, 5705, 2014.
(要約)
Since the establishment of embryonic stem (ES) cell lines, the combined use of gene targeting with homologous recombination has aided in elucidating the functions of various genes. However, the ES cell technique is inefficient and time-consuming. Recently, two new gene-targeting technologies have been developed: the transcription activator-like effector nuclease (TALEN) system, and the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein (Cas) system. In addition to aiding researchers in solving conventional problems, these technologies can be used to induce site-specific mutations in various species for which ES cells have not been established. Here, by targeting the Fgf10 gene through RNA microinjection in one-cell mouse embryos with the TALEN and CRISPR/Cas systems, we produced the known limb-defect phenotypes of Fgf10-deficient embryos at the F0 generation. Compared to the TALEN system, the CRISPR/Cas system induced the limb-defect phenotypes with a strikingly higher efficiency. Our results demonstrate that although both gene-targeting technologies are useful, the CRISPR/Cas system more effectively elicits single-step biallelic mutations in mice.
Y Uehara, Jun Hirose, S Yamabe, N Okamoto, T Okada, Seiichi Oyadomari and H Mizuta : Endoplasmic reticulum stress-induced apoptosis contributes to articular cartilage degeneration via C/EBP homologous protein., Osteoarthritis and Cartilage, Vol.22, No.7, 1007-1017, 2014.
(要約)
In vivo, the degree of ER stress was similar in Chop(-/-)- and Chop(+/+) mice. However, in Chop(-/-) mice apoptosis and cartilage degeneration were lower by 26.4% and 42.4% at 4 weeks, by 26.8% and 44.9% at 8 weeks, and by 26.9% and 32.3% at 12 weeks after surgery than Chop(+/+) mice, respectively. In vitro, the degree of ER stress induction by TM was similar in Chop(-/-)- and Chop(+/+) chondrocytes. On the other hand, apoptosis was 55.3% lower and the suppression of collagen type II and aggrecan mRNA was 21.0% and 23.3% less, and the increase of matrix metalloproteinase-13 mRNA was 20.0% less in Chop(-/-)- than Chop(+/+) chondrocytes.
S Yoshimoto, TM Loo, K Atarashi, H Kanda, S Sato, Seiichi Oyadomari, Y Iwakura, K Oshima, H Morita, M Hattori, K Honda, Y Ishikawa, E Hara and N Ohtani : Obesity-induced gut microbial metabolite promotes liver cancer through senescence secretome., Nature, Vol.499, No.7456, 97-101, 2013.
(要約)
Obesity has become more prevalent in most developed countries over the past few decades, and is increasingly recognized as a major risk factor for several common types of cancer. As the worldwide obesity epidemic has shown no signs of abating, better understanding of the mechanisms underlying obesity-associated cancer is urgently needed. Although several events were proposed to be involved in obesity-associated cancer, the exact molecular mechanisms that integrate these events have remained largely unclear. Here we show that senescence-associated secretory phenotype (SASP) has crucial roles in promoting obesity-associated hepatocellular carcinoma (HCC) development in mice. Dietary or genetic obesity induces alterations of gut microbiota, thereby increasing the levels of deoxycholic acid (DCA), a gut bacterial metabolite known to cause DNA damage. The enterohepatic circulation of DCA provokes SASP phenotype in hepatic stellate cells (HSCs), which in turn secretes various inflammatory and tumour-promoting factors in the liver, thus facilitating HCC development in mice after exposure to chemical carcinogen. Notably, blocking DCA production or reducing gut bacteria efficiently prevents HCC development in obese mice. Similar results were also observed in mice lacking an SASP inducer or depleted of senescent HSCs, indicating that the DCA-SASP axis in HSCs has key roles in obesity-associated HCC development. Moreover, signs of SASP were also observed in the HSCs in the area of HCC arising in patients with non-alcoholic steatohepatitis, indicating that a similar pathway may contribute to at least certain aspects of obesity-associated HCC development in humans as well. These findings provide valuable new insights into the development of obesity-associated cancer and open up new possibilities for its control.
K Morotomi-Yano, Seiichi Oyadomari, H Akiyama and KI. Yano : Nanosecond pulsed electric fields act as a novel cellular stress that induces translational suppression accompanied by eIF2 phosphorylation and 4E-BP1 dephosphorylation., Experimental Cell Research, Vol.318, No.14, 1733-1744, 2012.
(要約)
Recent advances in electrical engineering enable the generation of ultrashort electric fields, namely nanosecond pulsed electric fields (nsPEFs). Contrary to conventional electric fields used for DNA electroporation, nsPEFs can directly reach intracellular components without membrane destruction. Although nsPEFs are now recognized as a unique tool in life sciences, the molecular mechanism of nsPEF action remains largely unclear. Here, we present evidence that nsPEFs act as a novel cellular stress. Exposure of HeLa S3 cells to nsPEFs quickly induced phosphorylation of eIF2α, activation of its upstream stress-responsive kinases, PERK and GCN2, and translational suppression. Experiments using PERK- and GCN2-knockout cells demonstrated dual contribution of PERK and GCN2 to nsPEF-induced eIF2α phosphorylation. Moreover, nsPEF exposure yielded the elevated GADD34 expression, which is known to downregulate the phosphorylated eIF2α. In addition, nsPEF exposure caused a rapid decrease in 4E-BP1 phosphorylation irrespective of the PERK/GCN2 status, suggesting participation of both eIF2α and 4E-BP1 in nsPEF-induced translational suppression. RT-PCR analysis of stress-inducible genes demonstrated that cellular responses to nsPEFs are distinct from those induced by previously known forms of cellular stress. These results provide new mechanistic insights into nsPEF action and implicate the therapeutic potential of nsPEFs for stress response-associated diseases.
(キーワード)
Adaptor Proteins, Signal Transducing / Electricity / Eukaryotic Initiation Factor-2 / HeLa Cells / Humans / Phosphoproteins / Phosphorylation / Stress, Physiological / Time Factors
Chisayo Kozuka, Kouichi Yabiku, Sumito Sunagawa, Rei Ueda, Shin-Ichiro Taira, Hiroyuki Ohshiro, Tomomi Ikema, Ken Yamakawa, Moritake Higa, Hideaki Tanaka, Chitoshi Takayama, Masayuki Matsushita, Seiichi Oyadomari, Michio Shimabukuro and Hiroaki Masuzaki : Brown Rice and Its Component, γ-Oryzanol, Attenuate the Preference for High-Fat Diet by Decreasing Hypothalamic Endoplasmic Reticulum Stress in Mice., Diabetes, Vol.61, No.12, 3084-3093, 2012.
(要約)
Brown rice is known to improve glucose intolerance and prevent the onset of diabetes. However, the underlying mechanisms remain obscure. In the current study, we investigated the effect of brown rice and its major component, γ-oryzanol (Orz), on feeding behavior and fuel homeostasis in mice. When mice were allowed free access to a brown rice-containing chow diet (CD) and a high-fat diet (HFD), they significantly preferred CD to HFD. To reduce hypothalamic endoplasmic reticulum (ER) stress on an HFD, mice were administered with 4-phenylbutyric acid, a chemical chaperone, which caused them to prefer the CD. Notably, oral administration of Orz, a mixture of major bioactive components in brown rice, also improved glucose intolerance and attenuated hypothalamic ER stress in mice fed the HFD. In murine primary neuronal cells, Orz attenuated the tunicamycin-induced ER stress. In luciferase reporter assays in human embryonic kidney 293 cells, Orz suppressed the activation of ER stress-responsive cis-acting elements and unfolded protein response element, suggesting that Orz acts as a chemical chaperone in viable cells. Collectively, the current study is the first demonstration that brown rice and Orz improve glucose metabolism, reduce hypothalamic ER stress, and, consequently, attenuate the preference for dietary fat in mice fed an HFD.
L Andreas Birkenfeld, Hui-Young Lee, Sachin Majumdar, J Michael Juzcak, Joaopaul Camporez, R Francois Jornayvaz, W David Frederick, Blas Guigni, Mario Kahn, Dongyang Zhang, Dirk Weismann, M Ayman Arafat, F Andreas Pfeiffer, Steffanie Lieske, Seiichi Oyadomari, David Ron, T Varman Samuel and I Gerald Shulman : Influence of the hepatic eIF2{alpha} ER stress response pathway on insulin mediated ER stress, Hepatic and Peripheral Glucose Metabolism., The Journal of Biological Chemistry, 2011.
(要約)
Recent studies have implicated endoplasmic reticulum (ER) stress in insulin resistance associated with caloric excess. Surprisingly we did not detect activation of ER stress pathways in mice placed on a 3 day high fat diet (HFD), despite hepatic lipid accumulation and insulin resistance. However, infusion of insulin in a hyperinsulinemic euglycemic clamp induced conspicuous ER stress in the 3day HFD mice, which was mirrored by enhanced ER stress markers following exposure of primary hepatocytes to insulin in vitro. To clarify the role of the liver ER stress-dependent eIF2α-P pathway in response to acute caloric excess on liver and muscle glucose and lipid metabolism, we studied transgenic mice in which the hepatic ER stress-dependent eIF2α-P pathway was inhibited by over-expressing a constitutively active C-terminal fragment of GADD34/PPP1R15a (GC), a regulatory subunit of phosphatase that terminates ER stress signaling by phospho-eIF2α. Inhibition of the eIF2α-P signaling in liver led to a decrease in fasting plasma glucose concentration, which could be attributed to reduced hepatic glucose production in the basal and clamped state. In contrast, these mice were surprisingly found to have impaired insulin-stimulated muscle and adipose tissue insulin sensitivity. This latter effect could be attributed to an increase in circulating IGFBP-3 levels in the transgenic animals. These data imply that hepatic ER stress signaling is not requisite for hepatic insulin resistance and may be a consequence of hyperinsulinemia. Moreover, hepatic ER stress-dependent eIF2α-P signaling is implicated in an unanticipated cross-talk between the liver and peripheral organs to influences insulin sensitivity via IGBBP-3.
T Satoh, N Abiru, M Kobayashi, H Zhou, K Nakamura, G Kuriya, H Nakamura, Y Nagayama, E Kawasaki, H Yamasaki, L Yu, S G Eisenbarth, E Araki, M Mori, Seiichi Oyadomari and K Eguchi : CHOP deletion does not impact the development of diabetes but suppresses the early production of insulin autoantibody in the NOD mouse., Apoptosis, Vol.16, No.4, 438-448, 2011.
(要約)
C/EBP homologous protein (CHOP) has been proposed as a key transcription factor for endoplasmic reticulum (ER) stress-mediated -cell death induced by inflammatory cytokines in vitro. However, the contribution of CHOP induction to the pathogenesis of type 1 diabetes is not yet clear. To evaluate the relevance of CHOP in the pathogenesis of type 1 diabetes in vivo, we generated CHOP-deficient non-obese diabetic (NOD.Chop (-/-)) mice. CHOP deficiency did not affect the development of insulitis and diabetes and apoptosis in -cells. Interestingly, NOD.Chop (-/-) mice exhibited a delayed appearance of insulin autoantibodies compared to wild-type (wt) mice. Adoptive transfer with the diabetogenic, whole or CD8(+)-depleted splenocytes induced -cell apoptosis and the rapid onset of diabetes in the irradiated NOD.Chop (-/-) recipients with similar kinetics as in wt mice. Expression of ER stress-associated genes was not significantly up-regulated in the islets from NOD.Chop (-/-) compared to those from wt mice or NOD-scid mice. These findings suggest that CHOP expression is independent of the development of insulitis and diabetes but might affect the early production of insulin autoantibodies in the NOD mouse.
Keisuke Yamamoto, Kazuna Takahara, Seiichi Oyadomari, Tetsuya Okada, Takashi Sato, Akihiro Harada and Kazutoshi Mori : Induction of liver steatosis and lipid droplet formation in ATF6alpha-knockout mice burdened with pharmacological endoplasmic reticulum stress., Molecular Biology of the Cell, Vol.21, No.17, 2975-2986, 2010.
(要約)
Accumulation of unfolded proteins in the endoplasmic reticulum (ER) activates homeostatic responses collectively termed the unfolded protein response. Among the three principal signaling pathways operating in mammals, activating transcription factor (ATF)6alpha plays a pivotal role in transcriptional induction of ER-localized molecular chaperones and folding enzymes as well as components of ER-associated degradation, and thereby mouse embryonic fibroblasts deficient in ATF6alpha are sensitive to ER stress. However, ATF6alpha-knockout mice show no apparent phenotype under normal growing conditions. In this report, we burdened mice with intraperitoneal injection of the ER stress-inducing reagent tunicamycin and found that wild-type mice were able to recover from the insult, whereas ATF6alpha-knockout mice exhibited liver dysfunction and steatosis. Thus, ATF6alpha-knockout mice accumulated neutral lipids in the liver such as triacylglycerol and cholesterol, which was ascribable to blockage of beta-oxidation of fatty acids caused by decreased mRNA levels of the enzymes involved in the process, suppression of very-low-density lipoprotein formation due to destabilized apolipoprotein B-100, and stimulation of lipid droplet formation resulting from transcriptional induction of adipose differentiation-related protein. Accordingly, the hepatocytes of tunicamycin-injected knockout mice were filled with many lipid droplets. These results establish links among ER stress, lipid metabolism, and steatosis.
Yasuyo Ariyama, Yoshito Tanaka, Hiroyuki Shimizu, Kenju Shimomura, Shuichi Okada, Tsugumichi Saito, Eijiro Yamada, Seiichi Oyadomari, Masataka Mori and Masatomo Mori : The role of CHOP messenger RNA expression in the link between oxidative stress and apoptosis., Metabolism: Clinical and Experimental, Vol.57, No.12, 1625-1635, 2008.
(要約)
Low expression of antioxidant enzymes makes pancreatic beta-cells susceptible to cell damage by oxidative stress. Pancreatic beta-cell loss caused by endoplasmic reticulum stress is associated with the onset of diabetes mellitus. The present studies were undertaken to investigate a possible involvement of proapoptotic gene CHOP in pancreatic beta-cells damage by oxidative stress. The induction of CHOP messenger RNA and apoptosis were investigated in betaHC-9 cells after the oxidative stress by hydrogen peroxide and ribose. Latter was examined after the suppression of CHOP by small interfering RNA. For in vivo study, the pancreatic beta-cells were examined in CHOP-knockout (KO) mice after multiple low-dose streptozotocin (MLDS) administration. In betaHC-9 cells, both hydrogen peroxide and ribose obviously increased apoptotic cells, accompanied with enhanced CHOP messenger RNA expression. However, the number of apoptotic cells by those stimulations was significantly reduced by the addition of small interfering RNA against CHOP. In vivo study also showed that CHOP-KO mice were less susceptible to diabetes after MLDS administration. Although the oxidative stress marker level was similar to that of MLDS-treated wild type, the pancreatic beta-cell area was maintained in CHOP-KO mice. The present studies showed that CHOP should be important in pancreatic beta-cell injury by oxidative stress and indicate that CHOP may play a role in the development of pancreatic beta-cell damage on the onset of diabetes mellitus.
Kseniya Petrova, Seiichi Oyadomari, Linda M. Hendershot and David Ron : Regulated association of misfolded endoplasmic reticulum lumenal proteins with P58/DNAJc3., The EMBO Journal, Vol.27, No.21, 2862-2872, 2008.
(要約)
P58/DNAJc3 defends cells against endoplasmic reticulum (ER) stress. Most P58 molecules are translocated into the ER lumen, and here we report selective and stable binding to misfolded proteins by P58's TPR-containing N-terminal domain. In vitro, too, P58 binds selectively to a model misfolded protein and challenge of that complex with physiological concentrations of the ER lumenal Hsp70-type chaperone BiP encourages disassembly. BiP-induced dissociation of P58 from its substrate depends on the presence of ATP and on interactions with P58's J-domain, which are mediated by invariant residues BiP(R197) and P58(H422). A functional J-domain also accelerates dissociation of P58 from a model substrate, VSV-G(ts045), on the latter's re-folding in vivo. However, J-domain binding can be separated from the ability to promote substrate dissociation by the mutant BiP(E201G) and a wild-type J-domain fused ectopically to P58(H422Q) rescues the latter's inability to dissociate from substrate in response to BiP and ATP. These findings are consistent with a model whereby localized activation of the Hsp70-type partner is sufficient to promote substrate handover from the J-domain co-chaperone.
Seiichi Oyadomari, Heather P. Harding, Yuhong Zhang, Miho Oyadomari and David Ron : Dephosphorylation of translation initiation factor 2alpha enhances glucose tolerance and attenuates hepatosteatosis in mice., Cell Metabolism, Vol.7, No.6, 520-532, 2008.
(要約)
The molecular mechanisms linking the stress of unfolded proteins in the endoplasmic reticulum (ER stress) to glucose intolerance in obese animals are poorly understood. In this study, enforced expression of a translation initiation factor 2alpha (eIF2alpha)-specific phosphatase, GADD34, was used to selectively compromise signaling in the eIF2(alphaP)-dependent arm of the ER unfolded protein response in liver of transgenic mice. The transgene resulted in lower liver glycogen levels and susceptibility to fasting hypoglycemia in lean mice and glucose tolerance and diminished hepatosteatosis in animals fed a high-fat diet. Attenuated eIF2(alphaP) correlated with lower expression of the adipogenic nuclear receptor PPARgamma and its upstream regulators, the transcription factors C/EBPalpha and C/EBPbeta, in transgenic mouse liver, whereas eIF2alpha phosphorylation promoted C/EBP translation in cultured cells and primary hepatocytes. These observations suggest that eIF2(alphaP)-mediated translation of key hepatic transcriptional regulators of intermediary metabolism contributes to the detrimental consequences of nutrient excess.
Yasuyo Ariyama, Hiroyuki Shimizu, Tetsurou Satoh, Takafumi Tsuchiya, Shuichi Okada, Seiichi Oyadomari, Masataka Mori and Masatomo Mori : Chop-deficient mice showed increased adiposity but no glucose intolerance., Obesity, Vol.15, No.7, 1647-1656, 2007.
(要約)
OBJECTIVE: CCAAT/enhancer binding protein (C/EBP) homologous protein (CHOP)-10/growth arrest and DNA damage 153 is a dominant-negative member of the C/EBP transcription family and inhibits adipogenesis in vitro. The study was undertaken to determine the role of CHOP in obesity in vivo. RESEARCH METHODS AND PROCEDURES: Changes in daily food consumption and body weight were measured. The weight of white and brown adipose tissue was compared between Chop(+/+) and (-/-) mice fed normal chow or a high-fat diet. Glucose and insulin tolerance tests were done, and serum adipocytokine was measured to determine their metabolic state. Fat cell size of subcutaneous and mesenteric adipose tissue was microscopically observed. C/EBP expression in white adipose tissue was examined by Western blot. RESULTS: Female Chop(-/-) mice had significantly greater body weight and adiposity than Chop(+/+) mice, although daily food intake and rectal temperature did not differ. In comparison with Chop(+/+) mice, glucose tolerance and insulin sensitivity did not differ in female Chop(-/-) mice, but levels of plasma leptin and adiponectin were higher. High-fat diet feeding resulted in obesity in female Chop(+/-) and (-/-) mice, although caloric intake did not differ from that of Chop(+/+) mice. Fat cell area was larger in mesenteric fat but not in subcutaneous fat in Chop(-/-) mice fed a high-fat diet. C/EBPbeta and the 30-kDa form of C/EBPalpha expressions were increased in parametrial fat of Chop(-/-) mice, but the 42-kDa form of C/EBPalpha expression was lower than in Chop(+/+) mice. DISCUSSION: CHOP deficiency causes obesity in female animals without severe metabolic disorders, and C/EBP's expression may be considered to participate in the process.
(キーワード)
Animals / Body Weight / Glucose Tolerance Test / Mice / Mice, Knockout / Models, Animal / Obesity / Transcription Factor CHOP
Seiichi Oyadomari, Chi Yun, Edward A. Fisher, Nicola Kreglinger, Gert Kreibich, Miho Oyadomari, Heather P. Harding, Alan G. Goodman, Hanna Harant, Jennifer L. Garrison, Jack Taunton, Michael G. Katze and David Ron : Cotranslocational degradation protects the stressed endoplasmic reticulum from protein overload., Cell, Vol.126, No.4, 727-739, 2006.
(要約)
The ER's capacity to process proteins is limited, and stress caused by accumulation of unfolded and misfolded proteins (ER stress) contributes to human disease. ER stress elicits the unfolded protein response (UPR), whose components attenuate protein synthesis, increase folding capacity, and enhance misfolded protein degradation. Here, we report that P58(IPK)/DNAJC3, a UPR-responsive gene previously implicated in translational control, encodes a cytosolic cochaperone that associates with the ER protein translocation channel Sec61. P58(IPK) recruits HSP70 chaperones to the cytosolic face of Sec61 and can be crosslinked to proteins entering the ER that are delayed at the translocon. Proteasome-mediated cytosolic degradation of translocating proteins delayed at Sec61 is cochaperone dependent. In P58(IPK-/-) mice, cells with a high secretory burden are markedly compromised in their ability to cope with ER stress. Thus, P58(IPK) is a key mediator of cotranslocational ER protein degradation, and this process likely contributes to ER homeostasis in stressed cells.
Maiko Awai, Takahisa Koga, Yasuya Inomata, Seiichi Oyadomari, Tomomi Gotoh, Masataka Mori and Hidenobu Tanihara : NMDA-induced retinal injury is mediated by an endoplasmic reticulum stress-related protein, CHOP/GADD153., Journal of Neurochemistry, Vol.96, No.1, 43-52, 2005.
(要約)
We investigated the role of an endoplasmic reticulum stress-associated protein, CHOP/GADD153, after NMDA-induced mouse retinal damage. After injection of NMDA into the vitreous, TUNEL-positive cells were detected in the retinal ganglion cell layer (GCL) and inner nuclear layer (INL) at 6 h after NMDA injection, and these gradually increased in number up to 24 h. Analysis by real-time RT-PCR revealed that CHOP mRNA was induced by about 3-fold, at 2 h after NMDA injection. Immunoreactivity for the CHOP protein was intense in cells of the GCL following NMDA treatment. Immunoblot analysis showed that NMDA injection increased the expression of CHOP protein in the retina. Compared with wild-type mice, CHOP/ mice were more resistant to NMDA-induced retinal cell death as determined by TUNEL assay. At 7 days after NMDA treatment, the thickness of the inner plexiform layer and INL were larger in CHOP/ mice than in wild-type mice. The number of residual cells in the GCL following NMDA treatment was significantly higher in CHOP/ mice than in wild-type mice. In conclusion, CHOP is induced in mouse retina by NMDA treatment, and CHOP/ mice are more resistant to NMDA-induced retinal damage, suggesting that CHOP plays an important role in NMDA-induced retinal cell death.
Motoyoshi Endo, Seiichi Oyadomari, Moritaka Suga, Masataka Mori and Tomomi Gotoh : The ER stress pathway involving CHOP is activated in the lungs of LPS-treated mice., The Journal of Biochemistry, Vol.138, No.4, 501-507, 2005.
(要約)
CHOP is a C/EBP family transcription factor involved in endoplasmic reticulum (ER) stress-mediated apoptosis. To determine if the ER stress pathway is involved in the pathogenesis of LPS-treated mouse lung injury, mice were given lipopolysaccharide (LPS) intraperitoneally. The mRNAs for activating transcription factor (ATF) 4 and X-box binding protein (XBP) 1, transcriptional activators of the CHOP gene, and that for CHOP were induced by or after the LPS treatment. Apoptosis induced by LPS treatment was suppressed in the lungs of Chop-knockout mice. Overexpression of CHOP induced apoptosis in a lung cancer-derived cell line. These results suggest that the ER stress pathway, involving CHOP, is activated and plays a role in the pathogenesis of septic shock lung.
Stefan J. Marciniak, Chi Y. Yun, Seiichi Oyadomari, Isabel Novoa, Yuhong Zhang, Rivka Jungreis, Kazuhiro Nagata, Heather P. Harding and David Ron : CHOP induces death by promoting protein synthesis and oxidation in the stressed endoplasmic reticulum., Genes & Development, Vol.18, No.24, 3066-3077, 2004.
(要約)
Unfolded and malfolded client proteins impose a stress on the endoplasmic reticulum (ER), which contributes to cell death in pathophysiological conditions. The transcription factor C/EBP homologous protein (CHOP) is activated by ER stress, and CHOP deletion protects against its lethal consequences. We find that CHOP directly activates GADD34, which promotes ER client protein biosynthesis by dephosphorylating phospho-Ser 51 of the alpha-subunit of translation initiation factor 2 (eIF2alpha) in stressed cells. Thus, impaired GADD34 expression reduces client protein load and ER stress in CHOP(-/-) cells exposed to perturbations that impair ER function. CHOP(-/-) and GADD34 mutant cells accumulate less high molecular weight protein complexes in their stressed ER than wild-type cells. Furthermore, mice lacking GADD34-directed eIF2alpha dephosphorylation, like CHOP(-/-) mice, are resistant to renal toxicity of the ER stress-inducing drug tunicamycin. CHOP also activates ERO1alpha, which encodes an ER oxidase. Consequently, the ER of stressed CHOP(-/-) cells is relatively hypo-oxidizing. Pharmacological and genetic manipulations that promote a hypo-oxidizing ER reduce abnormal high molecular weight protein complexes in the stressed ER and protect from the lethal consequences of ER stress. CHOP deletion thus protects cells from ER stress by decreasing ER client protein load and changing redox conditions within the organelle.
David Ron and Seiichi Oyadomari : Lipid phase perturbations and the unfolded protein response., Developmental Cell, Vol.7, No.3, 287-288, 2004.
(要約)
Recent studies of the consequences of ganglioside accumulation in lysosomal storage disease and free cholesterol accumulation in cell membranes in atherosclerosis suggest an unexpected link between perturbation of the endoplasmic reticulum membrane's lipid phase, induction of the unfolded protein response, and cell death.
(キーワード)
Animals / Arteriosclerosis / Cell Death / Cell Membrane / Endoplasmic Reticulum / Humans / Lipid Metabolism / Lipids / Protein Denaturation / Protein Folding / Proteins
Seiichi Oyadomari and M Mori : Roles of CHOP/GADD153 in endoplasmic reticulum stress., Cell Death and Differentiation, Vol.11, No.4, 381-389, 2004.
(要約)
Endoplasmic reticulum (ER) is the site of synthesis and folding of secretory proteins. Perturbations of ER homeostasis affect protein folding and cause ER stress. ER can sense the stress and respond to it through translational attenuation, upregulation of the genes for ER chaperones and related proteins, and degradation of unfolded proteins by a quality-control system. However, when the ER function is severely impaired, the organelle elicits apoptotic signals. ER stress has been implicated in a variety of common diseases such as diabetes, ischemia and neurodegenerative disorders. One of the components of the ER stress-mediated apoptosis pathway is C/EBP homologous protein (CHOP), also known as growth arrest- and DNA damage-inducible gene 153 (GADD153). Here, we summarize the current understanding of the roles of CHOP/GADD153 in ER stress-mediated apoptosis and in diseases including diabetes, brain ischemia and neurodegenerative disease.
S Tajiri, Seiichi Oyadomari, S Yano, M Morioka, T Gotoh, J I. Hamada, Y Ushio and M Mori : Ischemia-induced neuronal cell death is mediated by the endoplasmic reticulum stress pathway involving CHOP., Cell Death and Differentiation, Vol.11, No.4, 403-415, 2004.
(要約)
Brain ischemia induces apoptosis in neuronal cells, but the mechanism is not well understood. When wild-type mice were subjected to bilateral common carotid arteries occlusion (BCCAO) for 15 min, apoptosis-associated morphological changes and appearance of TUNEL-positive cells were observed in the striatum and in the hippocampus at 48 h after occlusion. RT-PCR analysis revealed that mRNAs for ER stress-associated proapoptotic factor CHOP and an ER chaperone BiP are markedly induced at 12 h after BCCAO. Immunohistochemical analysis showed that CHOP protein is induced in nuclei of damaged neurons at 24 h after occlusion. In contrast, ischemia-associated apoptotic loss of neurons was decreased in CHOP(-/-) mice. Primary hippocampal neurons from CHOP(-/-) mice were more resistant to hypoxia-reoxygenation-induced apoptosis than those from wild-type animals. These results indicate that ischemia-induced neuronal cell death is mediated by the ER stress pathway involving CHOP induction.
T Gotoh, K Terada, Seiichi Oyadomari and M Mori : hsp70-DnaJ chaperone pair prevents nitric oxide- and CHOP-induced apoptosis by inhibiting translocation of Bax to mitochondria., Cell Death and Differentiation, Vol.11, No.4, 390-402, 2004.
(要約)
We reported that the endoplasmic reticulum (ER) stress pathway involving CHOP, a member of the C/EBP transcription factor family, plays a key role in nitric oxide (NO)-mediated apoptosis of macrophages and pancreatic beta cells. We also showed that the cytosolic chaperone pair of hsp70 and dj1 (hsp40/hdj-1) or dj2 (HSDJ/hdj-2) prevents NO-mediated apoptosis upstream of cytochrome c release from mitochondria. To analyze roles of the chaperone pair in preventing apoptosis, RAW 264.7 macrophages stably expressing hsp70 and dj1 or dj2 were established. The chaperone pair prevented LPS/IFN-gamma-induced and NO-mediated apoptosis downstream of CHOP induction. hsp70 mutant protein lacking the ATPase domain or the C-terminal EEVD sequence were not effective in preventing CHOP-induced apoptosis. A mutant dj2 lacking the C-terminal prenylation CaaX motif, was also not effective. When wild-type RAW 264.7 cells were treated with LPS/IFN-gamma, NO-mediated apoptosis was induced, and proapoptotic Bcl-2 family protein Bax was translocated from cytosol to mitochondria. This translocation was prevented in cells stably expressing hsp70/dj2, and in CHOP knockout cells. Overexpression of CHOP in wild-type cells also induced translocation of Bax and this translocation was prevented in cells expressing hsp70/dj2. CHOP-induced apoptosis was prevented by Bax knock-down. Coimmunoprecipitation experiments showed that Bax interacts with both hsp70 and dj1/dj2. ATPase domain of hsp70 was necessary for the binding with Bax. These findings indicate that CHOP-induced apoptosis is mediated by translocation of Bax from the cytosol to the mitochondria, and hsp70/dj1 or dj2 chaperone pair prevents apoptosis by interacting with Bax and preventing translocation to the mitochondria.
(キーワード)
Adenosine Triphosphatases / Animals / Apoptosis / CCAAT-Enhancer-Binding Proteins / COS Cells / Cell Line / Cercopithecus aethiops / Cytosol / HSP40 Heat-Shock Proteins / HSP70 Heat-Shock Proteins / Heat-Shock Proteins / Macrophages / Mice / Mice, Knockout / Mitochondria / Molecular Chaperones / Nitric Oxide / Protein Transport / Proto-Oncogene Proteins / Proto-Oncogene Proteins c-bcl-2 / Transcription Factor CHOP / Transcription Factors / Transfection / bcl-2-Associated X Protein
Kwang-Jong Lee, Kazutoyo Terada, Seiichi Oyadomari, Yukihiro Inomata, Masataka Mori and Tomomi Gotoh : Induction of molecular chaperones in carbon tetrachloride-treated rat liver: implications in protection against liver damage., Cell Stress & Chaperones, Vol.9, No.1, 58-68, 2004.
(要約)
Carbon tetrachloride (CCl4) induces liver damage, apparently through the formation of free-radical metabolites. Molecular chaperones such as heat shock protein (Hsp) of 70 kDa have been found to protect cells from various stresses. We previously found that cytosolic chaperone pairs of the Hsp70 family and their DnaJ homolog cochaperones prevent nitric oxide-mediated apoptosis and heat-induced cell death. Expression of cytosolic chaperones, including Hsp70; heat shock cognate (Hsc) 70; and DnaJ homologs dj1 (DjB1/Hsp40/hdj-1), dj2 (DjA1/HSDJ/hdj-2), dj3 (DjA2), and dj4 (DjA4), in the liver of CCl4-treated rats was analyzed. Messenger ribonucleic acids for all these chaperones were markedly induced 3-12 hours after CCl4 treatment with a maximum at 6 hours. Hsp70 and dj1 proteins were markedly induced at 6-24 hours with a maximum at 12 hours, whereas dj2 and dj4 were moderately induced at around 12 hours. Hsc70 was weakly induced after treatment, and dj3 was little induced. To better understand the significance of the induction of chaperones, the effect of preinduction of chaperones on CCl4-induced liver damage was analyzed. When chaperones were preinduced in the liver by heat treatment, increase in serum alanine aminotransferase activity after CCl4 treatment was significantly attenuated. Hsp90, another major cytosolic chaperone, also was induced by heat treatment. On the other hand, Mn- and Cu/Zn-superoxide dismutase were not induced by heat treatment or by CCl4 treatment. These results suggest that cytosolic chaperones of Hsp70 and DnaJ families or Hsp90 (or both) are induced in CCl4-treated rat liver to protect the hepatocytes from the damage being inflicted.
Céline Jousse, Seiichi Oyadomari, Isabel Novoa, Phoebe Lu, Yuhong Zhang, Heather P. Harding and David Ron : Inhibition of a constitutive translation initiation factor 2alpha phosphatase, CReP, promotes survival of stressed cells., The Journal of Cell Biology, Vol.163, No.4, 767-775, 2003.
(要約)
Phosphorylation of eukaryotic translation initiation factor 2alpha (eIF2alpha) on serine 51 is effected by specific stress-activated protein kinases. eIF2alpha phosphorylation inhibits translation initiation promoting a cytoprotective gene expression program known as the integrated stress response (ISR). Stress-induced activation of GADD34 feeds back negatively on this pathway by promoting eIF2alpha dephosphorylation, however, GADD34 mutant cells retain significant eIF2alpha-directed phosphatase activity. We used a somatic cell genetic approach to identify a gene encoding a novel regulatory subunit of a constitutively active holophosphatase complex that dephosphorylates eIF2alpha. RNAi of this gene, which we named constitutive repressor of eIF2alpha phosphorylation (CReP, or PPP1R15B), repressed the constitutive eIF2alpha-directed phosphatase activity and activated the ISR. CReP RNAi strongly protected mammalian cells against oxidative stress, peroxynitrite stress, and more modestly against accumulation of malfolded proteins in the endoplasmic reticulum. These findings suggest that therapeutic inhibition of eIF2alpha dephosphorylation by targeting the CReP-protein-phosphatase-1 complex may be used to access the salubrious qualities of the ISR.
Eiichi Araki, Seiichi Oyadomari and Masataka Mori : Impact of endoplasmic reticulum stress pathway on pancreatic beta-cells and diabetes mellitus., Experimental Biology and Medicine, Vol.228, No.10, 1213-1217, 2003.
(要約)
Diabetes is caused by impaired insulin secretion in pancreatic beta-cells and peripheral insulin resistance. Overload of pancreatic beta-cells leads to beta-cell exhaustion and finally to the development of diabetes. Reduced beta-cell mass is evident in type 2 diabetes, and apoptosis is implicated in this process. One characteristic feature of beta-cells is highly developed endoplasmic reticulum (ER) due to a heavy engagement in insulin secretion. The ER serves several important functions, including post-translational modification, folding, and assembly of newly synthesized secretory proteins, and its proper function is essential to cell survival. Various conditions can interfere with ER function and these conditions are called ER stress. Recently, we found that nitric oxide (NO)-induced apoptosis in beta-cells is mediated by the ER-stress pathway. NO causes ER stress and leads to apoptosis through induction of ER stress-associated apoptosis factor CHOP. The Akita mouse with a missense mutation (Cys96Tyr) in the insulin 2 gene has hyperglycemia and a reduced beta-cell mass. This mutation disrupts a disulfide bond between A and B chains of insulin and may induce its conformational change. In the development of diabetes in Akita mice, mRNAs for an ER chaperone Bip and CHOP were induced in the pancreas. Overexpression of the mutant insulin in mouse MIN6 beta-cells induced CHOP expression and led to apoptosis. Targeted disruption of the CHOP gene did not delay the onset of diabetes in the homozygous Akita mice, but it protected islet cells from apoptosis and delayed the onset of diabetes in the heterozygous Akita mice. We conclude that ER overload in beta-cells causes ER stress and leads to apoptosis via CHOP induction. These results highlight the importance of chronic ER stress in beta-cell apoptosis in type 2 diabetes, and suggest a new target to the management of the disease.
(キーワード)
Animals / アポトーシス (apoptosis) / Diabetes Mellitus, Type 1 / Diabetes Mellitus, Type 2 / 小胞体 (endoplasmic reticulum) / Humans / インスリン (insulin) / Islets of Langerhans / 一酸化窒素 (nitric oxide) / Protein Folding / Stress, Physiological
(文献検索サイトへのリンク)
● PubMed @ National Institutes of Health, US National Library of Medicine (PMID): 14610263
Motoyoshi Endo, Seiichi Oyadomari, Yasuhiro Terasaki, Motohiro Takeya, Moritaka Suga, Masataka Mori and Tomomi Gotoh : Induction of arginase I and II in bleomycin-induced fibrosis of mouse lung., American Journal of Physiology. Lung Cellular and Molecular Physiology, Vol.285, No.2, L313-21, 2003.
(要約)
Arginase, which hydrolyzes arginine to urea and ornithine, is a precursor for the synthesis of polyamines and proline, which is abundant in collagen. The supply of proline can be a crucial factor in the process of lung fibrosis. We investigated the induction of arginine metabolic enzymes in bleomycin-induced mouse lung fibrosis. Histological studies and quantification of lung hydroxyproline showed that lung fibrosis develops in up to 14 days after bleomycin treatment. Under these conditions, collagen I mRNA was induced gradually in up to 15 days, and the content of hydroxyproline reached a maximum at 10 days. Arginase I mRNA was undetectable before bleomycin treatment but was induced 5-10 days after this treatment. Arginase I protein was induced at 7 days and remained little changed for up to 10 days and decreased at 14 days. On the other hand, arginase II mRNA that was detectable before treatment was increased gradually for up to 10 days and decreased at 14 days. Arginase II protein began to increase at day 5, increased for up to 10 days, and was decreased at day 14. mRNAs for cationic amino acid transporter-2 and ornithine decarboxylase were induced in a manner similar to that seen with collagen I mRNA. Immunohistochemical analysis showed that arginase I is induced in macrophages, whereas arginase II is induced in various cell types, including macrophages and myofibroblasts, and roughly colocalizes with the collagen-specific chaperone heat shock protein 47. Our findings suggest that arginine metabolic enzymes play an important role in the development of lung fibrosis, at least in mice.
Eiichi Araki, Seiichi Oyadomari and Masataka Mori : Endoplasmic reticulum stress and diabetes mellitus., Internal Medicine, Vol.42, No.1, 7-14, 2003.
(要約)
Pancreatic beta-cells are strongly engaged in protein secretion and have highly developed endoplasmic reticulum (ER). Proper folding of polypeptide into a three-dimensional structure is essential for cellular function and protein malfolding can threaten cell survival. Various conditions can perturb the protein folding in the ER, which is collectively called ER stress. In order to adapt ER stress conditions, the cells respond in three distinct ways such as transcriptional induction of ER chaperones, translational attenuation, and ER-associated degradation (ERAD). However, when ER functions are severely impaired, the cell is eliminated by apoptosis via transcriptional induction of CHOP/GADD153, the activation of cJUN NH2-terminal kinase, and/or the activation of caspase-12. Recent studies have revealed that beta-cell is one of the most susceptible cells for ER stress, and ER stress-mediated apoptosis in beta-cells can be a cause of diabetes. A comprehensive understanding of the impact of the ER stress pathway in beta-cells and how it relates to the development of diabetes may contribute to provide new targets for the prevention and treatment of this disease.
Takahisa Koga, Wen Yi Zhang, Tomomi Gotoh, Seiichi Oyadomari, Hidenobu Tanihara and Masataka Mori : Induction of citrulline-nitric oxide (NO) cycle enzymes and NO production in immunostimulated rat RPE-J cells., Experimental Eye Research, Vol.76, No.1, 15-21, 2003.
(要約)
Nitric oxide (NO) has been implicated in many physiological and pathological conditions in the eyes. The induction of inducible NO synthase (iNOS) and NO production have been noted in immunostimulated retinal pigment epithelial (RPE) cells. Cellular NO production depends on the availability of arginine, a substrate for NOS. Arginine can be regenerated from citrulline, another product of the NOS reaction, by argininosuccinate synthetase and argininosuccinate lyase, forming the citrulline-NO cycle. When rat RPE-J cells were treated with interferon-gamma (IFNgamma), tumor necrosis factor-alpha (TNFalpha) and lipopolysaccharide (LPS), and expression of the citrulline-NO cycle enzymes and related enzymes was analyzed, iNOS and argininosuccinate synthetase were highly induced at both mRNA and protein levels. On the other hand, argininosuccinate lyase was not induced. Among other related enzymes and transporters, mRNA for cationic amino acid transporter (CAT)-1 was weakly induced, whereas those for CAT-2, arginase I and II, ornithine aminotransferase and ornithine decarboxylase remained little changed. NO was produced by cells after stimulation with TNFalpha, IFNgamma and LPS. The induction of iNOS mRNA and the production of NO by these immunostimulated cells was further enhanced by cAMP. NO was produced from citrulline as well as from arginine. Our findings indicate that in activated RPE-J cells citrulline-arginine recycling is important for NO production.
Seiichi Oyadomari, E Araki and M Mori : Endoplasmic reticulum stress-mediated apoptosis in pancreatic β-cells, Apoptosis, Vol.7, No.4, 335-345, 2002.
(要約)
Apoptotic cell death in pancreatic beta-cells is involved in the pathogenesis of diabetes. Signals from death receptors and DNA damage have been widely accepted as being triggers of apoptosis in beta-cells. Recent studies indicated that the endoplasmic reticulum (ER) can sense and transduce apoptotic signals. Various genetic and environmental stresses interfere with protein folding in the ER and induce ER stress. In mammals, ER stress transducer proteins IRE1, PERK and ATF6 activate both survival and apoptotic pathways. The former includes transcriptional induction of ER chaperones, translational attenuation, and ER-associated degradation (ERAD) while the latter includes transcriptional induction of CHOP/GADD153, the activation of cJUN NH(2)-terminal kinase, and the activation of caspase-12. A characteristic feature of beta-cells is the highly developed ER apparently due to a heavy engagement in insulin secretion. beta-cells are most susceptible to ER stress. The recent studies reviewed in this article revealed that ER stress-mediated apoptosis in beta-cells plays an important role in the development of diabetes.
Seiichi Oyadomari, Akio Koizumi, Kiyoshi Takeda, Tomomi Gotoh, Shizuo Akira, Eiichi Araki and Masataka Mori : Targeted disruption of the Chop gene delays endoplasmic reticulum stress-mediated diabetes., The Journal of Clinical Investigation, Vol.109, No.4, 525-532, 2002.
(要約)
Overload of pancreatic beta cells in conditions such as hyperglycemia, obesity, and long-term treatment with sulfonylureas leads to beta cell exhaustion and type 2 diabetes. Because beta cell mass declines under these conditions, apparently as a result of apoptosis, we speculated that overload kills beta cells as a result of endoplasmic reticulum (ER) stress. The Akita mouse, which carries a conformation-altering missense mutation (Cys96Tyr) in Insulin 2, likewise exhibits hyperglycemia and a reduced beta cell mass. In the development of diabetes in Akita mice, mRNAs for the ER chaperone Bip and the ER stress-associated apoptosis factor Chop were induced in the pancreas. Overexpression of the mutant insulin in mouse MIN6 beta cells induced Chop expression and led to apoptosis. Targeted disruption of the Chop gene delayed the onset of diabetes in heterozygous Akita mice by 8-10 weeks. We conclude that ER overload in beta cells causes ER stress and leads to apoptosis via Chop induction. Our findings suggest a new therapeutic approach for preventing the onset of diabetes by inhibiting Chop induction or by increasing chaperone capacity in the ER.
Tomomi Gotoh, Seiichi Oyadomari, Kazutoshi Mori and Masataka Mori : Nitric oxide-induced apoptosis in RAW 264.7 macrophages is mediated by endoplasmic reticulum stress pathway involving ATF6 and CHOP., The Journal of Biological Chemistry, Vol.277, No.14, 12343-12350, 2002.
(要約)
Excess nitric oxide (NO) induces apoptosis in some cell types including macrophages; however, the cascade of NO-mediated apoptosis is not fully understood. We investigated the initial steps of NO-mediated apoptosis in mouse macrophage-like RAW 264.7 cells. When cells were treated with bacterial lipopolysaccharide (LPS) plus interferon-gamma (IFN-gamma), NO-mediated apoptosis occurred. Under these conditions, p53 accumulation was not observed, indicating that DNA damage is not the main trigger of NO-mediated apoptosis. On the other hand, mRNA and protein for CHOP, a transcription factor known to be induced by endoplasmic reticulum (ER) stress, were induced. The CHOP induction by LPS/IFN-gamma treatment preceded cytochrome c release from mitochondria. In addition, p90ATF6, an ER membrane-bound transcription factor involved in ER stress response, was cleaved to its active soluble form p50ATF6, which was transported to nucleus and bound to the ER stress response element of the CHOP gene. In the luciferase reporter assay, both the CHOP-binding element of the Rous sarcoma virus long terminal repeat and ER stress response element of the CHOP gene were activated by LPS/IFN-gamma treatment. When RAW 264.7 cells or COS-7 cells were transfected with expression plasmids for CHOP, p90ATF6, or p50ATF6, cell death was observed. In addition, apoptosis induced by p50ATF6 was prevented by a CHOP dominant negative form as well as by an ATF6 dominant negative form, and LPS/IFN-gamma-induced apoptosis was prevented by the CHOP dominant negative form. Peritoneal macrophages from CHOP knockout mice showed resistance to NO-induced apoptosis. These results indicate that the ER stress pathway involving ATF6 and CHOP plays a key role in NO-mediated apoptosis in macrophages.
(キーワード)
Activating Transcription Factor 6 / Animals / Apoptosis / CCAAT-Enhancer-Binding Proteins / COS Cells / Cell Line / Cell Membrane / Cell Nucleus / Cells, Cultured / Cytochrome c Group / DNA Damage / DNA-Binding Proteins / Endoplasmic Reticulum / Genes, Dominant / Immunoblotting / Interferon-gamma / Lipopolysaccharides / Luciferases / Macrophages / Mice / Mice, Knockout / Nitric Oxide / Plasmids / Protein Binding / RNA, Messenger / Time Factors / Transcription Factor CHOP / Transcription Factors / Transcription, Genetic / Transfection / Tumor Suppressor Protein p53
K Kawahara, Seiichi Oyadomari, T Gotoh, S Kohsaka, H Nakayama and M Mori : Induction of CHOP and apoptosis by nitric oxide in p53-deficient microglial cells., FEBS Letters, Vol.506, No.2, 135-139, 2001.
(要約)
Excessive nitric oxide (NO) has been implicated in neurotoxicity after stresses such as ischemia. NO toxicity is generally thought to be mediated by the DNA damage-p53 pathway or mitochondrial dysfunction. We investigated the mechanism of NO toxicity by using murine microglial MG5 cells established from p53-deficient mice. When MG5 cells were exposed to bacterial lipopolysaccharide plus interferon-gamma, mRNA and protein for inducible NO synthase (iNOS) were markedly induced, and apoptosis occurred. Under these conditions, we found that mRNA and protein for CHOP/GADD153, a C/EBP family transcription factor which is involved in endoplasmic reticulum (ER) stress-induced apoptosis, are induced. iNOS mRNA was induced 2 h after treatment, whereas CHOP mRNA began to increase at 6 h with a time lag. CHOP mRNA was also induced by NO donors S-nitroso-N-acetyl-DL-penicillamine (SNAP) or NOC18, or a peroxynitrite generator 3-(4-morpholinyl)-sydnonimine hydrochloride (SIN-1). Bip/GRP78, an ER chaperone which is known to be induced by ER stress, was also induced by SNAP or SIN-1, indicating that NO causes ER stress. These results suggest that NO-induced apoptosis in MG5 cells occurs through the ER stress pathway involving CHOP, but is independent of p53.
K Kawahara, T Gotoh, Seiichi Oyadomari, A Kuniyasu, S Kohsaka, M Mori and H Nakayama : Nitric oxide inhibits the proliferation of murine microglial MG5 cells by a mechanism involving p21 but independent of p53 and cyclic guanosine monophosphate., Neuroscience Letters, Vol.310, No.2-3, 89-92, 2001.
(要約)
We investigated the effect of nitric oxide (NO) on the proliferation of microglial MG5 cells established from p53-deficient mice. Cells were treated with bacterial lipopolysaccharide and interferon-gamma, and expression of inducible NO synthase (iNOS) and p21/waf1, a cyclin-dependent kinase inhibitor protein which is a critical downstream effector of p53, was investigated by RNA blot and immunoblot analyses. iNOS mRNA was induced 2 h after treatment and increased with time up to 24 h. p21 mRNA was expressed at a low level in untreated cells and increased with a kinetics similar to that for iNOS mRNA. iNOS and p21 proteins were also induced. An NO donor SNAP induced p21 mRNA and protein. SNAP inhibited incorporation of [(3)H]thymidine in MG5 cells in a dose-dependent manner. 8-Bromo-cGMP neither induced p21 mRNA nor inhibited [(3)H]thymidine incorporation. These results suggest that NO inhibits the proliferation of MG5 cells by induction of p21, which occurs independent of p53 and cGMP.
Seiichi Oyadomari, K Takeda, M Takiguchi, T Gotoh, M Matsumoto, I Wada, S Akira, E Araki and M Mori : Nitric oxide-induced apoptosis in pancreatic beta cells is mediated by the endoplasmic reticulum stress pathway., Proceedings of the National Academy of Sciences of the United States of America, Vol.98, No.19, 10845-10850, 2001.
(要約)
Excessive nitric oxide (NO) production in cytokine-activated beta cells has been implicated in beta cell disruption in type 1 diabetes. beta cells are very vulnerable to NO-induced apoptosis. However, the mechanism underlying this phenomenon is unclear. Low concentrations of NO that lead to apoptosis apparently do not cause severe DNA damage in mouse MIN6 beta cells. CHOP, a C/EBP homologous protein that is induced by endoplasmic reticulum (ER) stress and plays a role in growth arrest and cell death, was induced by a NO donor, S-nitroso-N-acetyl-D,L-penicillamine (SNAP). SNAP increased cytosolic Ca(2+), and only agents depleting ER Ca(2+) induced CHOP expression and led to apoptosis, suggesting that NO depletes ER Ca(2+). Overexpression of calreticulin increased the Ca(2+) content of ER and afforded protection to cells against NO-mediated apoptosis. Furthermore, pancreatic islets from CHOP knockout mice showed resistance to NO. We conclude that NO depletes ER Ca(2+), causes ER stress, and leads to apoptosis. Thus, ER Ca(2+) stores are a new target of NO, and the ER stress pathway is a major mechanism of NO-mediated beta cell apoptosis.
K Kawahara, T Gotoh, Seiichi Oyadomari, M Kajizono, A Kuniyasu, K Ohsawa, Y Imai, S Kohsaka, H Nakayama and M Mori : Co-induction of argininosuccinate synthetase, cationic amino acid transporter-2, and nitric oxide synthase in activated murine microglial cells., Brain Research. Molecular Brain Research, Vol.90, No.2, 165-173, 2001.
(要約)
Nitric oxide (NO) produced by activated microglia has been implicated in many pathophysiological events in the brain including neurodegenerative diseases. Cellular NO production depends absolutely on the availability of arginine, a substrate of NO synthase (NOS). Murine microglial MG5 cells were treated with bacterial lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma), and expression of inducible NO synthase (iNOS) and arginine-supplying enzymes was investigated by RNA blot analysis. iNOS mRNA was strongly induced after treatment and reached a maximum at 6-12 h. mRNA for argininosuccinate synthetase (AS), a citrulline-arginine recycling enzyme, increased at 6 h and reached a maximum at 12 h. Immunoblot analysis showed that iNOS and AS proteins were also induced. In addition, mRNA encoding the cationic amino acid transporter-2 (CAT-2) was strongly induced shortly after treatment. Induction of mRNAs for iNOS, AS, and CAT-2 by LPS/IFN-gamma was also observed following stimulation of rat primary microglial cells. These results strongly suggest that both arginine transport by CAT-2 and citrulline-arginine recycling are important for high-output production of NO in activated microglial cells.
Seiichi Oyadomari, T Gotoh, K Aoyagi, E Araki, M Shichiri and M Mori : Coinduction of endothelial nitric oxide synthase and arginine recycling enzymes in aorta of diabetic rats., Nitric Oxide: Biology and Chemistry, Vol.5, No.3, 252-260, 2001.
(要約)
Decreased availability of arginine and impaired production of NO (nitric oxide) have been implicated in the development of endothelial dysfunction. Citrulline formed by the NOS reaction is recycled to arginine by the citrulline-NO cycle, which is composed of NOS, argininosuccinate synthetase (AS), and argininosuccinate lyase. Therefore, we investigated the alterations of these enzymes in the aorta of streptozotocin (STZ)-induced diabetic rats. eNOS and AS mRNAs were increased by three- to fourfold 1-2 weeks after STZ treatment and decreased at 4 weeks. AL mRNA was weakly induced. Induction of eNOS and AS proteins was also observed. Cationic amino acid transporter (CAT)-1 mRNA remained little changed, and CAT-2 mRNA was not detected. The plasma nitrogen oxide levels were increased 1-2 weeks after STZ treatment and decreased at 4 weeks. Transforming growth factor-beta1 (TGF-beta1) mRNA in the aorta was also induced. TGF-beta1 induced eNOS and AS mRNAs in human umbilical vein endothelial cells but inhibited the proliferation of HUVEC. These results indicate that eNOS and AS are coinduced in the aorta in early stages of STZ-induced diabetic rats and that the induction is mediated by TGF-beta1. The results also suggest that TGF-beta1 works antiatherogenically at early stages of diabetes by increasing NO production, whereas prolonged elevation of TGF-beta1 functions atherogenically by inhibiting endothelial cell growth.
T Kimura, S Chowdhury, T Tanaka, A Shimizu, K Iwase, Seiichi Oyadomari, T Gotoh, H Matsuzaki, M Mori, S Akira and M Takiguchi : CCAAT/enhancer-binding protein beta is required for activation of genes for ornithine cycle enzymes by glucocorticoids and glucagon in primary-cultured hepatocytes., FEBS Letters, Vol.494, No.1-2, 105-111, 2001.
(要約)
Transcription of genes for enzymes of the ornithine cycle is activated by hormones such as glucocorticoids and glucagon. Promoters and enhancers of several genes for the enzymes interact with the CCAAT/enhancer-binding protein (C/EBP) family of transcription factors, and C/EBPbeta has been suggested to mediate glucocorticoid response of the gene for arginase, the last enzyme of the cycle. To determine the contribution of C/EBPbeta to hormonal regulation of genes for ornithine cycle enzymes, we examined mice with targeted disruption of the C/EBPbeta gene. Induction of genes for the enzymes by intraperitoneal injection of dexamethasone and glucagon was almost intact in the liver of C/EBPbeta-deficient mice. On the other hand, in primary-cultured hepatocytes derived from C/EBPbeta-deficient mice, induction of genes for the first enzyme carbamylphosphate synthetase, as well as for arginase, in response to dexamethasone and/or glucagon was severely impaired. Therefore, C/EBPbeta is required for hormonal induction of the genes for ornithine cycle enzymes in primary-cultured hepatocytes, while the deficiency of C/EBPbeta is compensated for in vivo.
W Y. Zhang, T Gotoh, Seiichi Oyadomari and M Mori : Coinduction of inducible nitric oxide synthase and arginine recycling enzymes in cytokine-stimulated PC12 cells and high output production of nitric oxide., Brain Research. Molecular Brain Research, Vol.83, No.1-2, 1-8, 2000.
(要約)
Nitric oxide (NO) is involved in many physiological and pathological processes in the brain. NO is synthesized from arginine by nitric oxide synthase (NOS), and the citrulline generated as a by-product can be recycled to arginine by argininosuccinate synthetase (AS) and argininosuccinate lyase (AL) via the citrulline-NO cycle. When neuronal PC12 cells differentiated with nerve growth factor were treated with interferon-gamma (IFNgamma) and tumor necrosis factor-alpha (TNFalpha), iNOS and AS mRNAs and proteins were markedly induced, with AL mRNA and protein being weakly induced. Cationic amino acid transporter-1 and -2 were not induced. IFNgamma or TNFalpha alone was ineffective. A large amount of NO (190 microM NO(2)(-) plus NO(3)(-) in culture medium in 24 h) was produced from arginine by cytokine-stimulated cells, and arginine could be replaced by citrulline. iNOS induction and NO production were attenuated by dexamethasone and dibutyryl cAMP and even more strongly so when combined. Therefore, a large amount of NO is produced in cytokine-stimulated PC12 cells following to induction of iNOS and citrulline-arginine recycling is important for NO production.
Seiichi Oyadomari, F Matsuno, S Chowdhury, T Kimura, K Iwase, E Araki, M Shichiri, M Mori and M Takiguchi : The gene for hepatocyte nuclear factor (HNF)-4alpha is activated by glucocorticoids and glucagon, and repressed by insulin in rat liver., FEBS Letters, Vol.478, No.1-2, 141-146, 2000.
(要約)
The gene for a transcription factor hepatocyte nuclear factor-4alpha (HNF-4alpha) is responsible for maturity-onset diabetes of the young, type 1. We examined hormonal regulation of the HNF-4alpha gene in the liver. Stimulation of primary-cultured rat hepatocytes with dexamethasone or glucagon led to induction of HNF-4alpha mRNA, being antagonized by insulin. In the liver of streptozotocin-induced diabetic rat, mRNA and protein levels for HNF-4alpha were elevated, and were normalized by insulin treatment. Therefore, HNF-4alpha in the liver is likely to be involved in the regulation of glucose metabolism in response to these hormones.
Junhong Gao, Yasushi Ishigaki, Tetsuya Yamada, Keiichi Kondo, Suguru Yamaguchi, Junta Imai, Kenji Uno, Yutaka Hasegawa, Shojiro Sawada, Hisamitsu Ishihara, Seiichi Oyadomari, Masataka Mori, Yoshitomo Oka and Hideki Katagiri : Involvement of Endoplasmic Stress Protein C/EBP Homologous Protein in Arteriosclerosis Acceleration With Augmented Biological Stress Responses, Circulation, Vol.124, No.7, 830-839, 2011.
(要約)
The processes of arteriosclerosis, including atherosclerosis and vascular remodeling, are affected by interactions among numerous biological pathways such as responses to inflammation, oxidative stress, and endoplasmic reticulum stress. C/EBP homologous protein (CHOP), which is well known to induce cellular apoptosis in response to severe endoplasmic reticulum stress, is reportedly upregulated in plaque lesions. We examined the effects of CHOP deficiency on 2 types of arteriosclerosis: cuff injury-induced neointimal formation and hypercholesterolemia-induced atherosclerosis. Cuff injury-induced neointimal formation was markedly inhibited in CHOP(-/-) mice with suppressed aortic expression of inflammatory factors and smooth muscle cell proliferation-related proteins. A CHOP deficiency also inhibited aortic plaque formation in hypercholesterolemic apolipoprotein E(-/-) mice with suppressed aortic expression of inflammatory factors and oxidative stress markers. Bone marrow transplantation experiments revealed that recipient CHOP deficiency significantly suppressed both cuff injury-induced neointimal formation and hypercholesterolemia-induced atherosclerotic plaque formation to a greater extent than donor CHOP deficiency, suggesting the importance of CHOP in vascular cells for arteriosclerosis progression. Furthermore, in our in vitro experiments, in not only macrophages but also endothelial and smooth muscle cell lines, endoplasmic reticulum stress inducers upregulated inflammation-, adhesion-, or smooth muscle cell proliferation-related proteins, whereas decreased CHOP expression remarkably suppressed endoplasmic reticulum stress-induced upregulation of these proteins. In addition to the well-known signaling for apoptosis induction, CHOP may play important roles in augmenting potentially pathological biological stress responses. This noncanonical role of CHOP, especially that expressed in vascular cells, may contribute to the progression of vascular remodeling and atherosclerosis.
Organs do not independently coordinate their metabolic activity:close communication between different organ systems is essential to regulate metabolism effectively. In recent years, the unfolded protein response(UPR), which is an adaptive mechanism to decrease the amount of unfolded or misfolded proteins in the ER, has been found to regulate metabolic function not only at the cellular level but also at the whole-organism level by way of inter-organ communications. This manuscript will present the most recent findings on the role of the UPR in inter-organ metabolic networks.
(キーワード)
小胞体 (endoplasmic reticulum) / Humans / Metabolic Networks and Pathways / Proteins / Unfolded Protein Response
(文献検索サイトへのリンク)
● PubMed @ National Institutes of Health, US National Library of Medicine (PMID): 30374012
Kaoru Yoshinaga, Akihiro Yasue, Seiichi Oyadomari and Eiji Tanaka : In vivo elucidation of the effect on the number of teeth caused by double mutant of WNT10A and WNT10B., 9th IOC, Yokohama, Oct. 2020.
Masato Miyake, Zhang Jun and Seiichi Oyadomari : Preemptive activation of integrated stress responses in adipose tissue suppresses food intake and improves obesity through growth and differentiation factor 15, EMBO workshop, Nov. 2019.
4.
Akira Izutani, Yuji Furumoto, Yoshimasa Hamada, Masato Miyake, Kenji Teranishi, Naoyuki Shimomura and Seiichi Oyadomari : The Influence of Applying High Electrical Field Pulses on Unfolded Protein Response of cells, The 2019 IEEE Pulsed Power and Plasma Science Conference, 4-pages, Orlando, Jun. 2019.
5.
Keisuke Kitakaze, Kiyoe Kurahashi, Masato Miyake, Yoshimasa Hamada, Miho Oyadomari and Seiichi Oyadomari : Targeted Deletion of ATF4 in β-cells Leads to the Vulnerability and Dedifferentiation During ER Stress, American Diabetes Association 78th scientific sessions, Jun. 2018.
6.
Yuji Furumoto, Daiki Sato, Yoshifumi Hamada, Masato Miyake, Kenji Teranishi, Naoyuki Shimomura and Seiichi Oyadomari : Activation of Endoplasmic Reticulum Stress Response by Applying of Nanosecond Pulsed Electric Fields for Medical Application, Proceedings of the 2018 IEEE International Power Modulator and High Voltage Conference, 456-460, Jackson, Jun. 2018.
Masatoshi Morimoto, Fumitake Tezuka, Fumio Hayashi, Kazuta Yamashita, Yoichiro Takata, Toshinori Sakai, Kousaku Higashino, Akihiro Nagamachi, Koichi Sairyo, Ryosuke Sato, Masato Miyake and Seiichi Oyadomari : ER STRESS AGGRAVATES THE HYPERTROPHY OF THE LIGAMNTUM FLAVUM, International Society for the Study of Lumbar Spine(ISSLS) 2017( May 29June 2, 2017), Athens, May 2017.
8.
Akihiro Yasue, Silvia Naomi Mitsui Akagi, Daishi Arai, Seiichi Oyadomari, Keita Sato, Junji Inoue, Seiichi Oyadomari and Eiji Tanaka : In vivo DNA deletion assay of MSX1 gene in mice using CRISPR/Cas system, JOINT MEETING of the German and Japanese Societies of Developmental Biologists, Kiel, Germany, Mar. 2017.
9.
Akihiro Yasue, Silvia Naomi Mitsui Akagi, Daishi Arai, Seiichi Oyadomari and Eiji Tanaka : In vivo DNA deleiton assay of Msx1 gene in mice using CRISPR/Cas system, Joint Meeting of the German and Japanese Societies of Developmental Biologists, Kiel, Germany, Mar. 2017.
10.
Seiichi Oyadomari : UPR-regulated miR-23a protects against pancreatic cell failure, Novel Forum, Karolinska Instututet, Stockholm, Sep. 2016.
11.
Silvia Naomi Mitsui Akagi, Akihiro Yasue, Kiyoshi Masuda, Takuya Naruto, Seiichi Oyadomari, Sumihare Noji, Issei Imoto and Eiji Tanaka : Conserved C-terminal domain of MSX1 is essential for tooth development, 12th Tooth Morphogenesis and Differentiation conference, Porvoo, Finland, Jun. 2016.
12.
Kiyoe Kurahashi, 森 智子, 宮本 千伸, 津川 和江, Miho Oyadomari, Kazuna Takahara, 木村 千寿子, Masato Miyake, Toshio Matsumoto and Seiichi Oyadomari : Saturated Fatty Acids Predominantly Activate PERK Pathway via Altered Composition of the Endoplasmic Reticulum Membrane, and Reduce Insulin Secretion in Pancreatic Cell by Translation Attenuation, 75th ADA scientific sessions, Jun. 2015.
13.
Hiroshi Fukuda, Masato Miyake, Hiroto Hirai, Kenji Teranishi, Naoyuki Shimomura and Seiichi Oyadomari : Effects on Endoplasmic Reticulum Stress Response of Applying Nanosecond Pulsed Electric Fields, Digest of Technical Papers-IEEE International Pulsed Power Conference, 370-373, Austin, Jun. 2015.
Akihiro Yasue, Silvia Naomi Mitsui Akagi, Teppei Watanabe, T Sakuma, Seiichi Oyadomari, T Yamamoto, Sumihare Noji, Taro Mito and Eiji Tanaka : Highly efficient targeted mutagenesis in one-cell mouse embryos mediated by TALEN and CRISPR/Cas systems, X meeting for Spanish Society for Developmental Biology (SEBD), Madrid, Oct. 2014.
15.
Masato Miyake, Kiyoe Kurahashi and Seiichi Oyadomari : Identification and Characterization of a Small-molecule Inducer of ATF4 for Promoting Insulin Synthesis in Pancreatic cells, 74th ADA Scientific Sessions, Jun. 2014.
16.
Hiroto Hirai, Masato Miyake, Akihiko Nagano, Kenji Teranishi, Naoyuki Shimomura and Seiichi Oyadomari : Investigation About Effects on Endoplasmic Reticulum Stress Responses by Applying Nanosecond Pulsed Electricfields, Proceedings of the 2014 IEEE International Power Modulator and High Voltage Conference, 419-422, Santa Fe, Jun. 2014.
Akihiro Yasue, Silvia Naomi Mitsui Akagi, H Watanabe, T Sakuma, Seiichi Oyadomari, T Yamamoto, Sumihare Noji, Taro Mito and Eiji Tanaka : A high efficient gene targeting in one-cell mouse embryos mediated by TALEN and CRISPR/Cas system., International Symposium on RNAi and Genome Editing Methods, Tokushima, Mar. 2014.
18.
Nomura Akitoshi, Masato Miyake, Atsushi Ogura, Kiyoe Kurahashi, Mori Tomoko, Tsugawa Kazue, Miyamoto Chinobu, Miho Oyadomari and Seiichi Oyadomari : Role of the PERK signaling pathway in metabolic process, International Symposium Hannover-Tokushima Research Communication, Aug. 2013.
19.
Seiichi Oyadomari : Role of Endoplasmic Reticulum Stress Response in Metabolic Regulation., International Symposium Hannover-Tokushima Research Communication, Aug. 2013.
20.
KOZUKA CHISAYO, YABIKU KOUICHI, SUNAGAWA SUMITO, UEDA REI, TAIRA SHIN-ICHIRO, YAMAKAWA KEN, HIGA MORITAKE, TAKAYAMA CHITOSHI, MATSUSHITA MASAYUKI, Seiichi Oyadomari, Michio Shimabukuro and MASUZAKI HIROAKI : Gamma-Oryzanol, a Unique Component of Brown Rice, Improves Glucose Metabolism in Mice, The American Diabetes Association's 73nd Scientific Sessions, Jul. 2013.
21.
Masato Miyake, NOMURA AKITOSHI, Atsushi Ogura, Kazuna Takahara, Kiyoe Kurahashi, Ryosuke Sato, Miho Oyadomari, Hiroshi Inoue and Seiichi Oyadomari : Phosphorylation of eIF2 in skeletal muscle increases energy expenditure and are resistant to diet induced obesity., The American Diabetes Association's 73nd Scientific Sessions, Jul. 2013.
22.
Chisayo Kozuka, Kouichi Yabiku, Chitoshi Takayama, Masayuki Matsushita, Seiichi Oyadomari, Michio Shimabukuro and Hiroaki Masuzaki : Gamma-oryzanol, a major component of brown rice, improves feeding behavior by decreasing hypothalamic endoplasmic reticulum stress in mice., 第90回 日本生理学会, Tokyo, Mar. 2013.
23.
Kazuna Takahara, R Murahashi, K Mori, Seiichi Oyadomari and Eiji Tanaka : ATF6 links endoplasmic reticulum (ER) stress to intestinal inflammation in mice., International Joint Symposium: The University of Tokushima, Universitas Gadjah Mada, Niigata University, Denpasar, Bali, Dec. 2010.
24.
Seiichi Oyadomari : The ER stress response as a possible link between metabolism and circadian rhythm., The 3rd International Symposium on Protein Community ISPC-Nara 2010, Sep. 2010.
25.
Seiichi Oyadomari : The ER stress response as a possible link between metabolism and circadian rhythm, Cell Stress Society International The 4th Internationa Congress on Stress Response in Biology and Metabolism, Sapporo Medical University School of Medicine, Oct. 2009.
荒井 大志, 泰江 章博, ミツイ アカギ シルビア ナオミ, 市原 亜起, 沢田 正樹, 親泊 政一, 田中 栄二 : Functional verification of C-terminal domain of Msx1 gene in mice for craniofacial development., 第42回日本分子生物学会年会, 2019年12月.
15.
Silvia Naomi Mitsui Akagi, Akihiro Yasue, Shinya Horiuchi, Seiichi Oyadomari and Eiji Tanaka : Rogdi plays an important role during bone and enamel development, 第78回日本矯正歯科学会学術大会: 日本矯正歯科学会大会プログラム・抄録集 78回 Page283.(2019), 283, Nov. 2019.
Silvia Naomi Mitsui Akagi, Akihiro Yasue, Shinya Horiuchi, Seiichi Oyadomari and Eiji Tanaka : Rogdi plays an important role during enamel mineralization., 第59回日本先天異常学会学術集会, Jul. 2019.
Habuta Munenori, Akihiro Yasue, Suzuki T Ken-ichi, Fujita Hirofumi, Bando Tetsuya, Sato Keita, Seiichi Oyadomari, Eiji Tanaka and Hideyo Ohuchi : Higher amount of the Fgf10 gene product is required for the accessory lobe formation and type 2 alveolar cell differentiation in the mouse lung as revealed by Fgf10-mosaic mutants generated by genome-editing., 第59回日本先天異常学会学術集会, Jul. 2019.
Silvia Naomi Mitsui Akagi, Akihiro Yasue, Shinya Horiuchi, Seiichi Oyadomari and Eiji Tanaka : CRISPR/Cas9-mediated targeting for the analysis of ROGDI in enamel mineralization., 第65回国際歯科研究学会日本部会総会・学術大会 JADR, Nov. 2017.
40.
Akihiro Yasue, Daishi Arai, Silvia Naomi Mitsui Akagi, Seiichi Oyadomari and Eiji Tanaka : In vivo deletion assay of mouse MSX1 gene using CRISPR/Cas system., 第65回国際歯科研究学会日本部会総会・学術大会 JADR, Nov. 2017.
41.
Silvia Naomi Mitsui Akagi, Akihiro Yasue, Shinya Horiuchi, Seiichi Oyadomari and Eiji Tanaka : CRISPR/Cas9-mediaated targeting for the analysis of ROGDI in enamel mineralization, The 65th Annual Meeting of Japanese Association for Dental Research, Nov. 2017.
42.
Akihiro Yasue, Daishi Arai, Silvia Naomi Mitsui Akagi, Seiichi Oyadomari and Eiji Tanaka : In vivo deletion assay of mouse MSX1 gene using CRISPR/Cas system, The 65th Annual Meeting of Japanese Association for Dental Research, Nov. 2017.
Silvia Naomi Mitsui Akagi, Akihiro Yasue, Shinya Horiuchi, Seiichi Oyadomari and Eiji Tanaka : The role of Rogdi in enamel biomineralization., 第76回日本矯正歯科学会学術大会, Oct. 2017.
Akihiro Yasue, Daishi Arai, Silvia Naomi Mitsui Akagi, Seiichi Oyadomari and Eiji Tanaka : Functional verification of each Msx homology domain of Msx1 gene for tooth morphogenesis using CRISPR/Cas system., 第50回日本発生生物学会, May 2017.
55.
Silvia Naomi Mitsui Akagi, Akihiro Yasue, Shinya Horiuchi, Seiichi Oyadomari and Eiji Tanaka : Rogdi is required for enamel biomineralization, 第50回日本発生生物学会, May 2017.
Silvia Naomi Mitsui Akagi, Akihiro Yasue, Issei Imoto, Seiichi Oyadomari and Eiji Tanaka : Biological validation of tooth agenesis causing mutation using CRISPR/Cas system in mice., The 75th Annual Meeting of the Japanese Orthodontic Society, Nov. 2016.
Akihiro Yasue, Silvia Naomi Mitsui Akagi, Teppei Watanabe, T Sakuma, Seiichi Oyadomari, T Yamamoto, Sumihare Noji, Taro Mito and Eiji Tanaka : A high efficient gene targeting in one-cell mouse embryos mediated by TALEN and CRISPR/Cas system, 第36回日本分子生物学会, Dec. 2013.
91.
Akihiro Yasue, Silvia Naomi Mitsui Akagi, Teppei Watanabe, T Sakuma, Seiichi Oyadomari, T Yamamoto, Sumihare Noji, Taro Mito and Eiji Tanaka : A high efficient gene targeting in one-cell mouse embryos mediated by TALEN and CRISPR/Cas system, 第36回日本分子生物学会, Dec. 2013.
Atsushi Ogura and Seiichi Oyadomari : Novel role of PERK-regulated non-coding RNAs in the unfolded protein response revealed by comprehensive gene expression analysis, The 24th CDB meeting, Jun. 2013.
10.
親泊 政一 : 糖尿病を発症させるアミロイドの蓄積・分解因子の網羅的探索, Proteolysis in the Regulation of Biological Processes 2010度班会議, 2010年11月.
Seiichi Oyadomari : Molecular mechanism of endoplasmic reticulum stress response, OMCC Seminar Series The 88th Cancer Therapeutics and Cardiotoxicity, Feb. 2009.