Mazaki Yuichi
| Faculty of Medicine Physiological Science Pharmacology | Lecturer |
Last Updated :2025/06/07
■Researcher basic information
Researchmap personal page
Researcher number
- 60311304
J-Global ID
Research Field
Educational Organization
- Bachelor's degree program, Departments of Medicine, School of Medicine
- Master's degree program, Graduate School of Medicine
- Doctoral (PhD) degree program, Graduate School of Medicine
■Career
Career
- 2015 - Present
Hokkaido University, Graduate School of Medicine, Lecture - 2013 - 2015
University of Occupational and Environmental Health, School of Medicine, Lecture - 2008 - 2013
Kumamoto University, Priority Organization for Innovation and Excellence, Special-appointment Assistant Professor - 2007 - 2008
Osaka Bioscience Institute, 1st Department, Research Scientist
■Research activity information
Papers
- Possible involvement of α, β-unsaturated carbonyl compounds in ferroptosis induced by the cigarette smoke extract of heated tobacco products in vascular smooth muscle cells.
Takahiro Horinouchi, Yuichi Mazaki, Soichi Miwa
Journal of pharmacological sciences, 158, 1, 8, 12, May 2025, [Peer-reviewed], [Domestic magazines]
English, Scientific journal, In this study, we aimed to determine the cytotoxic factors involved in ferroptosis induced by nicotine- and tar-free cigarette smoke extract (CSE) from heated tobacco products (HTPs) in vascular smooth muscle cells. CSE decreased mitochondrial metabolic activity and increased lactate dehydrogenase leakage. These cytotoxic effects completely disappeared after removing the carbonyls from the mainstream smoke. α, β-Unsaturated carbonyl compounds (acrolein, methyl vinyl ketone, crotonaldehyde, and methacrolein) in the mainstream smoke of HTPs and CSE caused cell damage, which was inhibited by a ferroptosis inhibitor, UAMC-3203. These results suggest that α, β-unsaturated carbonyl compounds might be involved in CSE-induced ferroptosis. - Phosphorylation of annexin A2 at serine 25 is required for endothelin-1 stimulated cell proliferation and AKT activation in melanoma cells
Yuichi Mazaki, Takahiro Horinouchi, Yasuhito Onodera, Jin-Min Nam
Biochemical and Biophysical Research Communications, 743, 151168, 151168, Elsevier BV, Jan. 2025, [Peer-reviewed], [Lead author, Corresponding author]
Scientific journal - Mechanism of cytotoxicity induced by the cigarette smoke extract (CSE) of heated tobacco products in vascular smooth muscle cells: A comparative study of the cytotoxic effects of CSE and the ferroptosis inducer, erastin
Takahiro Horinouchi, Yuichi Mazaki, Soichi Miwa
Journal of Pharmacological Sciences, Elsevier BV, Dec. 2023, [Peer-reviewed]
Scientific journal - LRRK2 is involved in the chemotaxis of neutrophils and differentiated HL-60 cells, and the inhibition of LRRK2 kinase activity increases fMLP-induced chemotactic activity.
Yuichi Mazaki, Haruka Handa, Yoshizuki Fumoto, Takahiro Horinouchi, Yasuhito Onodera
Cell communication and signaling : CCS, 21, 1, 300, 300, 30 Oct. 2023, [Peer-reviewed], [Lead author, Corresponding author], [International Magazine]
English, Scientific journal, BACKGROUND: Neutrophils depend heavily on glycolysis for energy production under normal conditions. In contrast, neutrophils require energy supplied by mitochondrial oxidative phosphorylation (OXPHOS) during chemotaxis. However, the mechanism by which the energy supply changes from glycolysis to OXPHOS remains unknown. Leucine-rich repeat kinase 2 (LRRK2) is partially present in the outer mitochondrial membrane fraction. Lrrk2-deficient cells show mitochondrial fragmentation and reduced OXPHOS activity. We have previously reported that mitofusin (MFN) 2 is involved in chemotaxis and OXPHOS activation upon chemoattractant N-formyl-Met-Leu-Phe (fMLP) stimulation in differentiated HL-60 (dHL-60) cells. It has been previously reported that LRRK2 binds to MFN2 and partially colocalizes with MFN2 at the mitochondrial membranes. This study investigated the involvement of LRRK2 in chemotaxis and MFN2 activation in neutrophils and dHL-60 cells. METHODS: Lrrk2 knockout neutrophils and Lrrk2 knockdown dHL-60 cells were used to examine the possible involvement of LRRK2 in chemotaxis. Lrrk2 knockdown dHL-60 cells were used a tetracycline-inducible small hairpin RNA (shRNA) system to minimize the effects of LRRK2 knockdown during cell culture. The relationship between LRRK2 and MFN2 was investigated by measuring the GTP-binding activity of MFN2 in Lrrk2 knockdown dHL-60 cells. The effects of LRRK2 kinase activity on chemotaxis were examined using the LRRK2 kinase inhibitor MLi-2. RESULTS: fMLP-induced chemotactic activity was reduced in Lrrk2 knockout neutrophils in vitro and in vivo. Lrrk2 knockdown in dHL-60 cells expressing Lrrk2 shRNA also reduced fMLP-induced chemotactic activity. Lrrk2 knockdown dHL-60 cells showed reduced OXPHOS activity and suppressed mitochondrial morphological change, similar to Mfn2 knockdown dHL-60 cells. The amount of LRRK2 in the mitochondrial fraction and the GTP-binding activity of MFN2 increased upon fMLP stimulation, and the MFN2 GTP-binding activity was suppressed in Lrrk2 knockdown dHL-60 cells. Furthermore, the kinase activity of LRRK2 and Ser935 phosphorylation of LRRK2 were reduced upon fMLP stimulation, and LRRK2 kinase inhibition by MLi-2 increased the migration to fMLP. CONCLUSIONS: LRRK2 is involved in neutrophil chemotaxis and the GTP-binding activity of MFN2 upon fMLP stimulation. On the other hand, the kinase activity of LRRK2 shows a negative regulatory effect on fMLP-induced chemotactic activity in dHL-60 cells. Video Abstract. - Cigarette smoke extract and its cytotoxic factor acrolein inhibit nitric oxide production in human vascular endothelial cells.
Takahiro Horinouchi, Yuichi Mazaki, Koji Terada, Soichi Miwa
Biological & pharmaceutical bulletin, 43, 11, 1804, 1809, Sep. 2020, [Peer-reviewed], [Domestic magazines]
English, Scientific journal, Acrolein (ACR), a highly reactive α,β-unsaturated aldehyde, is a major cytotoxic factor in nicotine- and tar-free cigarette smoke extract (CSE). There are conflicting results regarding endothelial functions despite the fact that both CSE and ACR cause cellular damage. Several lines of evidence indicate that CSE impairs endothelium-derived nitric oxide (NO)-dependent vasodilation by reducing the activity and protein expression of endothelial NO synthase (eNOS), whereas ACR elicits endothelium-dependent vasorelaxation by increasing the production of NO and expression of eNOS. To clarify whether CSE and its cytotoxic factor ACR cause endothelial dysfunction, this study examined the effects of CSE and ACR on human vascular endothelial EA.hy926 cells. CSE and ACR reduced the phosphorylation of eNOS at Ser1177 and total expression of eNOS. The CSE- and ACR-induced decrease in the phosphorylation and expression of eNOS was counteracted by glutathione (reduced form), an antioxidant. Basal NO production was inhibited by CSE, ACR, NG-nitro-L-arginine methyl ester (a competitive eNOS inhibitor), and nominally Ca2+-free solution supplemented with BAPTA-AM (a membrane permeable Ca2+ chelator). These results indicate that CSE and ACR increase oxidative stress, and reduce NO production by reducing the activity and total protein level of eNOS. - Extracellular Ca2+ promotes nitric oxide production via Ca2+-sensing receptor-Gq/11 protein-endothelial nitric oxide synthase signaling in human vascular endothelial cells.
Takahiro Horinouchi, Yuichi Mazaki, Koji Terada, Soichi Miwa
Journal of pharmacological sciences, 143, 4, 315, 319, Aug. 2020, [Peer-reviewed], [Domestic magazines]
English, Scientific journal, This study examined the possible involvement of Ca2+-sensing receptor (CaSR) in nitric oxide (NO) production in human vascular endothelial cells. Extracellular Ca2+ elevated the intracellular Ca2+ concentration, the endothelial NO synthase (eNOS) phosphorylation level, and NO release from the cells. These responses were inhibited by a CaSR antagonist and a Gq/11 protein inhibitor. Application of an endothelial cell suspension induced vasorelaxation in isolated rat thoracic aorta precontracted by phenylephrine. Adding an NO scavenger to the organ bath abolished this vasorelaxation response. These results suggest that extracellular Ca2+ promotes NO generation via CaSR- and Gq/11 protein-mediated eNOS activation. - Mitofusin 2 is involved in chemotaxis of neutrophil-like differentiated HL-60 cells.
Yuichi Mazaki, Shingo Takada, Junko Nio-Kobayashi, Satoshi Maekawa, Tsunehito Higashi, Yasuhito Onodera, Hisataka Sabe
Biochemical and biophysical research communications, 513, 3, 708, 713, Elsevier {BV}, 04 Jun. 2019, [Peer-reviewed], [Lead author, Corresponding author], [International Magazine]
English, Scientific journal, Neutrophils rapidly migrate to infection sites after the recognition of invaders. During chemotaxis, neutrophils require energy supplied by mitochondria oxidative phosphorylation (OXPHOS), whereas neutrophils rely heavily on glycolysis under normal conditions. Mitochondrial OXPHOS correlates with mitochondrial morphology. Here, we examined the mitochondrial morphology of neutrophil-like differentiated HL-60 cells after chemoattractant N-formyl-Met-Leu-Phe (fMLP) stimulation. We found that mitochondrial morphology changes to a tubular form after fMLP stimulation. Mitochondrial OXPHOS activity and mitochondrial complex II significantly increased after fMLP stimulation. On the other hand, the silencing of mitochondrial fusion protein mitofusin 2 (MFN2) suppresses mitochondrial morphological changes. Furthermore, MFN2 silencing suppressed OXPHOS activation and chemotaxis after fMLP stimulation. These results suggest that MFN2 is involved in chemotaxis of differentiated HL-60 cells depending on mitochondria. - Ca2+ signal is involved in endothelin-1-induced internalization of endothelin type A receptor expressed in Chinese hamster ovary cells.
Takahiro Horinouchi, Sarita Karki, Koji Terada, Yuichi Mazaki, Soichi Miwa
Journal of pharmacological sciences, 140, 1, 102, 105, May 2019, [Peer-reviewed], [Domestic magazines]
English, Scientific journal, Endothelin type A receptor (ETAR) is internalized upon agonist stimulation; however, the mechanism thereof remains controversial. In this study, we characterized the endothelin-1 (ET-1)-induced internalization of ETAR expressed in Chinese hamster ovary cells. ET-1 elicited ETAR internalization and increase in intracellular Ca2+ concentration. ET-1-induced ETAR internalization was completely inhibited by a reduction in intracellular and extracellular Ca2+ levels and partially suppressed by inhibitors of protein kinase C (PKC) and extracellular signal-regulated kinases 1/2 (ERK1/2), both of which are downstream molecules in ETAR signaling. These results suggest that Ca2+ mobilization, PKC, and ERK1/2 are involved in ET-1-induced ETAR internalization. - Annexin A2 is involved in activation of extracellular signal-regulated kinase upon endothelin-1 stimulation.
Yuichi Mazaki, Tsunehito Higashi, Takahiro Horinouchi, Soichi Miwa
Biochemical and biophysical research communications, 511, 1, 69, 72, Elsevier {BV}, 26 Mar. 2019, [Peer-reviewed], [Lead author, Corresponding author], [International Magazine]
English, Scientific journal, The overexpression of endothelin (ET)-1 or ET receptors (ETRs) is related to initiation and progression of tumor. In cancer cells, ET-1 activates various signaling pathways, including mitogen-activated protein kinase, phosphatidylinositol 3-kinase, protein kinase C through ETRs, although the mechanisms by which ET-1 activates these signaling pathways remain uncertain. Here, we found that ETRs interacted with annexin A2, which is overexpressed in various cancers. Annexin A2 bound to ET type A receptor and ET type B receptor. Upon ET-1 stimulation, serine phosphorylation of annexin A2 increased, while there is no change in tyrosine phosphorylation of annexin A2. On the other hand, annexin A2 silencing suppressed activation of ERK upon ET-1 stimulation. These results suggest that interaction of ETRs and annexin A2 may play important roles in activation of extracellular signal-regulated kinase upon ET-1 stimulation. - Endothelin type B receptor interacts with the 78-kDa glucose-regulated protein.
Yuichi Mazaki, Tsunehito Higashi, Yasuhito Onodera, Jin-Min Nam, Ari Hashimoto, Shigeru Hashimoto, Takahiro Horinouchi, Soichi Miwa
FEBS letters, 593, 6, 644, 651, Wiley, Mar. 2019, [Peer-reviewed], [Lead author, Corresponding author], [International Magazine]
English, Scientific journal, Endothelin (ET)-1 is involved in the vascular system, cell proliferation and apoptosis. ET receptors consist of ET type A receptor (ETA R) and ET type B receptor (ETB R). ETA R and ETB R generally exhibit opposite responses, although many exceptions exist. In the present study, we attempted to identify ETA R- or ETB R-specific binding proteins to understand the differences in ETA R- and ETB R-mediated responses after ET-1 stimulation. The 78-kDa glucose-regulated protein (GRP78) showed a stronger binding affinity towards ETB R than towards ETA R. Moreover, GRP78 overexpression promoted ETB R-mediated ERK activation and GRP78 silencing suppressed ETB R-mediated ERK activation. Furthermore, ETB R can localize GRP78 to the cell periphery. These results suggest that the interaction of ETB R with GRP78 affects ERK activation and GRP78 localization. - Glutathione and cysteines suppress cytotoxicity of gas phase of cigarette smoke by direct reacting with unsaturated carbonyl compounds in the gas phase.
Tsunehito Higashi, Enas Elmeligy, Yosuke Mai, Yoichi Noya, Koji Terada, Yuichi Mazaki, Yuji Kuge, Soichi Miwa
Biochemical and biophysical research communications, 509, 4, 988, 993, 19 Feb. 2019, [Peer-reviewed], [International Magazine]
English, Scientific journal, Unsaturated carbonyl compounds, such as acrolein (ACR) and methyl vinyl ketone (MVK), are environmental pollutants, and are contained in smoke, automobile exhaust, and heated oil. We have previously reported that major cytotoxic factors in the gas phase of cigarette smoke are ACR and MVK. ACR and MVK induce cell damage by reactive oxygen species generation via protein kinase C and NADPH oxidases, and antioxidants, such as glutathione (GSH) and N-acetylcysteine (NAC), can effectively suppress their cytotoxic activities. In this study, we attempted to elucidate the molecular mechanism(s) for suppression of ACR- and MVK-induced cytotoxic activities by these antioxidants. GSH, NAC, L- and D-cysteines completely suppressed cell damage induced by gas phase extract of cigarette smoke. The results of HPLC and mass spectrometry showed that GSH and NAC directly reacted with ACR and MVK. Cysteines and cysteine derivatives suppressed ACR-induced GAPDH carbonylation, a representative protein for carbonylation. The current results suggest that GSH, NAC, and cysteines directly reacted with ACR and MVK, and suppressed these unsaturated carbonyl compounds-induced cell damage by inhibition of protein carbonylation. - Chinese herbal medicine Qing-Dai-induced pulmonary arterial hypertension in a patient with ulcerative colitis: A case report and experimental investigation.
Kazuki Sato, Hiroshi Ohira, Takahiro Horinouchi, Toshitaka Nakaya, Yuichi Mazaki, Ayako Sugimoto, Taku Watanabe, Ichizo Tsujino, Masaharu Nishimura
Respiratory medicine case reports, 26, 265, 269, Elsevier {BV}, 2019, [Peer-reviewed], [International Magazine]
English, A recent case report described a case of pulmonary arterial hypertension (PAH) associated with use of the Chinese herbal medicine Qing-Dai; however, the clinical course and possible mechanisms have not been characterized. We present the case of a man with ulcerative colitis who was diagnosed with idiopathic PAH. After initiating oral beraprost therapy, the patient showed significant hemodynamic improvements and an unusual course of clinical recovery. In 2016, the Japanese Ministry of Health, Labour, and Welfare issued a warning regarding the possible side effects of Qing-Dai. We learned that our patient had been taking self-purchased Qing-Dai for 2 years. Therefore, we performed an experimental study and determined that Qing-Dai may cause PAH through a mechanism involving nitric oxide synthase inhibition and pulmonary artery endothelial dysfunction. - Protein kinase C-dependent cell damage by unsaturated carbonyl compounds in vascular cells.
Tsunehito Higashi, Yosuke Mai, Yuichi Mazaki
Journal of bioscience and bioengineering, 126, 4, 527, 532, Elsevier B.V., Oct. 2018, [Peer-reviewed], [Corresponding author], [Domestic magazines]
English, Scientific journal - Molecular mechanism for ET-1-induced insulin resistance in skeletal muscle cells
Takahiro Horinouchi, Yuichi Mazaki, Koji Terada, Soichi Miwa
Folia Pharmacologica Japonica, 151, 4, 140, 147, Japanese Pharmacological Society, 2018, [Peer-reviewed], [Invited], [Domestic magazines]
Japanese, Insulin resistance is a condition where the sensitivity to insulin of the tissues expressing insulin receptor (InsR) is decreased due to a functional disturbance of InsR-mediated intracellular signaling. Insulin promotes the entry of glucose into the tissues and skeletal muscle is the most important tissue responsible for the insulin's action of decreasing blood glucose levels. Endothelin-1 (ET-1), a potent vasoconstrictor and pro-inflammatory peptide, induces insulin resistance through a direct action on skeletal muscle. However, the signaling pathways of ET-1-induced insulin resistance in skeletal muscle remain unclear. Here we show molecular mechanism underlying the inhibitory effect of ET-1 on insulin-stimulated Akt phosphorylation and glucose uptake in myotubes of rat L6 skeletal muscle cell line. mRNA expression levels of differentiation marker genes, MyoD and myogenin, were increased during L6 myoblasts differentiation into myotubes. Some of myotubes possessed the ability to spontaneously contract. In myotubes, insulin promoted Akt phosphorylation at Thr308 and Ser473, and [3H]-labelled 2-deoxy-D-glucose ([3H]2-DG) uptake. The insulin-facilitated Akt phosphorylation and [3H]2-DG uptake were inhibited by ET-1. The inhibitory effect of ET-1 was counteracted by blockade of ET type A receptor (ETAR), inhibition of Gq/11 protein, and siRNA knockdown of G protein-coupled receptor kinase 2 (GRK2). The exogenously overexpressed GRK2 directly bound to endogenous Akt and their association was facilitated by ET-1. In summary, activation of ETAR with ET-1 inhibits insulin-induced Akt phosphorylation and [3H]2-DG uptake in a Gq/11 protein- and GRK2-dependent manner in skeletal muscle. These findings indicate that ETAR and GRK2 are potential targets for insulin resistance. - Intracellular Ca2+ is an essential factor for cell damage induced by unsaturated carbonyl compounds.
Tsunehito Higashi, Yosuke Mai, Yuichi Mazaki, Soichi Miwa
Journal of bioscience and bioengineering, 124, 6, 680, 684, Dec. 2017, [Peer-reviewed], [Domestic magazines]
English, Scientific journal - ARF1 recruits RAC1 to leading edge in neutrophil chemotaxis.
Yuichi Mazaki, Yasuhito Onodera, Tsunehito Higashi, Takahiro Horinouchi, Tsukasa Oikawa, Hisataka Sabe
Cell communication and signaling : CCS, 15, 1, 36, 36, 02 Oct. 2017, [Peer-reviewed], [Lead author, Corresponding author], [International Magazine]
English, Scientific journal - Endothelin-1 suppresses insulin-stimulated Akt phosphorylation and glucose uptake via GPCR kinase 2 in skeletal muscle cells.
Takahiro Horinouchi, Akimasa Hoshi, Takuya Harada, Tsunaki Higa, Sarita Karki, Koji Terada, Tsunehito Higashi, Yosuke Mai, Prabha Nepal, Yuichi Mazaki, Soichi Miwa
British journal of pharmacology, 173, 6, 1018, 32, Mar. 2016, [Peer-reviewed], [International Magazine]
English, Scientific journal - Carbonyl Compounds in the Gas Phase of Cigarette Mainstream Smoke and Their Pharmacological Properties.
Takahiro Horinouchi, Tsunehito Higashi, Yuichi Mazaki, Soichi Miwa
Biological & pharmaceutical bulletin, 39, 6, 909, 14, 2016, [Peer-reviewed], [Domestic magazines]
English, Scientific journal - A Standardized Method for the Preparation of a Gas Phase Extract of Cigarette Smoke.
Tsunehito Higashi, Yosuke Mai, Yuichi Mazaki, Takahiro Horinouchi, Soichi Miwa
Biological & pharmaceutical bulletin, 39, 6, 898, 902, 2016, [Peer-reviewed], [Domestic magazines]
English, Scientific journal - Overexpression of Peroxiredoxin 4 Affects Intestinal Function in a Dietary Mouse Model of Nonalcoholic Fatty Liver Disease.
Aya Nawata, Hirotsugu Noguchi, Yuichi Mazaki, Toshihiro Kurahashi, Hiroto Izumi, Ke-Yong Wang, Xin Guo, Hidetaka Uramoto, Kimitoshi Kohno, Hatsumi Taniguchi, Yoshiya Tanaka, Junichi Fujii, Yasuyuki Sasaguri, Akihide Tanimoto, Toshiyuki Nakayama, Sohsuke Yamada
PloS one, 11, 4, e0152549, 2016, [Peer-reviewed], [International Magazine]
English, Scientific journal - The dynamics of mucosal-associated invariant T cells in multiple sclerosis.
Chie Sugimoto, Makoto Hirotani, Kazunori Yoshikiyo, Uichi Koshimizu, Rika Wakao, Takahiro Horinouchi, Yuichi Mazaki, Tsunehiko Higashi, Toshiyuki Fukazawa, Hiroyoshi Fujita, Hidenao Sasaki, Hiroshi Wakao
SpringerPlus, 5, 1, 1259, 1259, 2016, [Peer-reviewed], [International Magazine]
English, Scientific journal - GBF1 bears a novel phosphatidylinositol-phosphate binding module, BP3K, to link PI3Kγ activity with Arf1 activation involved in GPCR-mediated neutrophil chemotaxis and superoxide production.
Yuichi Mazaki, Yasuharu Nishimura, Hisataka Sabe
Molecular biology of the cell, 23, 13, 2457, 67, Jul. 2012, [Peer-reviewed], [Lead author, Corresponding author], [International Magazine]
English, Scientific journal - Fbx8 makes Arf6 refractory to function via ubiquitination.
Hajime Yano, Itaru Kobayashi, Yasuhito Onodera, Frédéric Luton, Michel Franco, Yuichi Mazaki, Shigeru Hashimoto, Kazuhiro Iwai, Ze'ev Ronai, Hisataka Sabe
Molecular biology of the cell, 19, 3, 822, 32, Mar. 2008, [Peer-reviewed], [International Magazine]
English, Scientific journal - GEP100 links epidermal growth factor receptor signalling to Arf6 activation to induce breast cancer invasion.
Masaki Morishige, Shigeru Hashimoto, Eiji Ogawa, Yoshinobu Toda, Hirokazu Kotani, Mayumi Hirose, Shumei Wei, Ari Hashimoto, Atsuko Yamada, Hajime Yano, Yuichi Mazaki, Hiroshi Kodama, Yoshinori Nio, Toshiaki Manabe, Hiromi Wada, Hidenori Kobayashi, Hisataka Sabe
Nature cell biology, 10, 1, 85, 92, Jan. 2008, [Peer-reviewed], [International Magazine]
English, Scientific journal - CIN85, a Cbl-interacting protein, is a component of AMAP1-mediated breast cancer invasion machinery.
Jin-Min Nam, Yasuhito Onodera, Yuichi Mazaki, Hiroyuki Miyoshi, Shigeru Hashimoto, Hisataka Sabe
The EMBO journal, 26, 3, 647, 56, 07 Feb. 2007, [Peer-reviewed], [International Magazine]
English, Scientific journal - ArfGAP family proteins in cell adhesion, migration and tumor invasion.
Hisataka Sabe, Yasuhito Onodera, Yuichi Mazaki, Shigeru Hashimoto
Current opinion in cell biology, 18, 5, 558, 64, Oct. 2006, [Peer-reviewed], [International Magazine]
English, Scientific journal - Neutrophil direction sensing and superoxide production linked by the GTPase-activating protein GIT2.
Yuichi Mazaki, Shigeru Hashimoto, Tohru Tsujimura, Masaki Morishige, Ari Hashimoto, Kosuke Aritake, Atsuko Yamada, Jin-Min Nam, Hiroshi Kiyonari, Kazuki Nakao, Hisataka Sabe
Nature immunology, 7, 7, 724, 31, Jul. 2006, [Peer-reviewed], [International Magazine]
English, Scientific journal - Expression of AMAP1, an ArfGAP, provides novel targets to inhibit breast cancer invasive activities.
Yasuhito Onodera, Shigeru Hashimoto, Ari Hashimoto, Masaki Morishige, Yuichi Mazaki, Atsuko Yamada, Eiji Ogawa, Masashi Adachi, Takaki Sakurai, Toshiaki Manabe, Hiromi Wada, Nariaki Matsuura, Hisataka Sabe
The EMBO journal, 24, 5, 963, 73, 09 Mar. 2005, [Peer-reviewed], [International Magazine]
English, Scientific journal - Roles played by a subset of integrin signaling molecules in cadherin-based cell-cell adhesion.
Hajime Yano, Yuichi Mazaki, Kazuo Kurokawa, Steven K Hanks, Michiyuki Matsuda, Hisataka Sabe
The Journal of cell biology, 166, 2, 283, 95, 19 Jul. 2004, [Peer-reviewed], [International Magazine]
English, Scientific journal - Localized suppression of RhoA activity by Tyr31/118-phosphorylated paxillin in cell adhesion and migration.
Asako Tsubouchi, Junko Sakakura, Ryohei Yagi, Yuichi Mazaki, Erik Schaefer, Hajime Yano, Hisataka Sabe
The Journal of cell biology, 159, 4, 673, 83, 25 Nov. 2002, [Peer-reviewed], [International Magazine]
English, Scientific journal - Paxillin associates with poly(A)-binding protein 1 at the dense endoplasmic reticulum and the leading edge of migrating cells.
Alison J Woods, Marnie S Roberts, Jyoti Choudhary, Simon T Barry, Yuichi Mazaki, Hisataka Sabe, Simon J Morley, David R Critchley, Jim C Norman
The Journal of biological chemistry, 277, 8, 6428, 37, 22 Feb. 2002, [Peer-reviewed], [International Magazine]
English, Scientific journal - PAG3/Pap alpha/KIAA0400, a GTPase-activating protein for ADP-ribosylation factor (ARF), regulates ARF6 in Fc gamma receptor-mediated phagocytosis of macrophages
H Uchida, A Kondo, Y Yoshimura, Y Mazaki, H Sabe
JOURNAL OF EXPERIMENTAL MEDICINE, 193, 8, 955, 966, Apr. 2001, [Peer-reviewed]
English, Scientific journal - An ADP-ribosylation factor GTPase-activating protein Git2-short/KIAA0148 is involved in subcellular localization of paxillin and actin cytoskeletal organization
Y Mazaki, S Hashimoto, K Okawa, A Tsubouchi, K Nakamura, R Yagi, H Yano, A Kondo, A Iwamatsu, A Mizoguchi, H Sabe
MOLECULAR BIOLOGY OF THE CELL, 12, 3, 645, 662, Mar. 2001, [Peer-reviewed]
English, Scientific journal - Interaction of paxillin with p21-activated kinase (PAK). Association of paxillin α with the kinase-inactive and the Cdc42-activated forms of PAK3
Shigeru Hashimoto, Asako Tsubouchi, Yuichi Mazaki, Hisataka Sabe
Journal of Biological Chemistry, 276, 8, 6037, 6045, American Society for Biochemistry {\&} Molecular Biology ({ASBMB}), 23 Feb. 2001, [Peer-reviewed]
English, Scientific journal - A new paxillin-binding protein, PAG3/Pap alpha/KIAA0400, bearing an ADP-ribosylation factor GTPase-activating protein activity, is involved in paxillin recruitment to focal adhesions and cell migration
A Kondo, S Hashimoto, H Yano, K Nagayama, Y Mazaki, H Sabe
MOLECULAR BIOLOGY OF THE CELL, 11, 4, 1315, 1327, Apr. 2000, [Peer-reviewed]
English, Scientific journal - A truncated isoform of the PP2A B56 subunit promotes cell motility through paxillin phosphorylation
A Ito, TR Kataoka, M Watanabe, K Nishiyama, Y Mazaki, H Sabe, Y Kitamura, H Nojima
EMBO JOURNAL, 19, 4, 562, 571, Feb. 2000, [Peer-reviewed]
English, Scientific journal - Paxillin isoforms in mouse - Lack of the gamma isoform and developmentally specific beta isoform expression
Y Mazaki, H Uchida, O Hino, S Hashimoto, H Sabe
JOURNAL OF BIOLOGICAL CHEMISTRY, 273, 35, 22435, 22441, Aug. 1998, [Peer-reviewed]
English, Scientific journal - Role of Mitf in differentiation and transdifferentiation of chicken pigmented epithelial cell
M Mochii, Y Mazaki, N Mizuno, H Hayashi, G Eguchi
DEVELOPMENTAL BIOLOGY, 193, 1, 47, 62, Jan. 1998, [Peer-reviewed]
English, Scientific journal - Mitosis specific serine phosphorylation and downregulation of one of the focal adhesion protein, paxillin
R Yamaguchi, Y Mazaki, K Hirota, S Hashimoto, H Sabe
ONCOGENE, 15, 15, 1753, 1761, Oct. 1997, [Peer-reviewed]
English, Scientific journal - Monocyte cells and cancer cells express novel paxillin isoforms with different binding properties to focal adhesion proteins
Y Mazaki, S Hashimoto, H Sabe
JOURNAL OF BIOLOGICAL CHEMISTRY, 272, 11, 7437, 7444, Mar. 1997, [Peer-reviewed]
English, Scientific journal - Role of integrins in differentiation of chick retinal pigmented epithelial cells in vitro
Y Mazaki, M Mochii, R Kodama, G Eguchi
DEVELOPMENT GROWTH & DIFFERENTIATION, 38, 4, 429, 437, Aug. 1996, [Peer-reviewed], [Domestic magazines]
English
Other Activities and Achievements
- Role of LRRK2 on neutrophil chemotaxis upon fMLP stimulation
真崎雄一, 半田悠, 麓佳月, 日本分子生物学会年会プログラム・要旨集(Web), 46th, 2023 - Roles of MFN2 and MFN2 associated protein in chemotaxis of neutrophil-like differentiated HL-60 cells
Mazaki Yuichi, Higashi Tsunehito, Nio-Kobayashi Junko, Onodera Yasuhito, Proceedings for Annual Meeting of The Japanese Pharmacological Society, 96, 2-B-P-112, 2022
Neutrophils are important in innate immunity and in the initiation of an acute response to infection. Under normal conditions, the mitochondrial membrane potential of neutrophils is low, and neutrophils energy depends fundamentally on glycolysis. In contrast, neutrophils require energy supplied from mitochondrial oxidative phosphorylation (OXPHOS) during infection. The inhibition of mitochondrial OXPHOS blocks the chemotaxis of neutrophils. Here, we examined the mitochondrial morphology of neutrophil-like differentiated HL-60 cells after chemoattractant N-formyl-Met-Leu-Phe (fMLP) stimulation. We found that mitochondrial morphology changes to a tubular form after fMLP stimulation. Mitochondrial OXPHOS activity and mitochondrial complex II significantly increased after fMLP stimulation. On the other hand, the silencing of mitochondrial fusion protein mitofusin 2 (MFN2) suppresses mitochondrial morphological changes. MFN2 silencing suppressed OXPHOS activation and chemotaxis after fMLP stimulation. Furthermore, the silencing of MFN2 associated protein suppresses also mitochondrial morphological changes and chemotaxis upon fMLP stimulation. These results suggest that MFN2 and MFN2 associated protein are involved in chemotaxis of differentiated HL-60 cells., Japanese Pharmacological Society, Japanese - Carbonyl compounds in the gas phase extract of mainstream smoke derived from heat-not-burn and combustion cigarettes cause vascular endothelial dysfunction.
Horinouchi Takahiro, Mazaki Yuichi, Miwa Soichi, Proceedings for Annual Meeting of The Japanese Pharmacological Society, 95, 2-P-143, 2022
The gas phase extract of mainstream smoke of combustion cigarettes includes many carbonyl compounds, which increase oxidative stress. The present study examined whether carbonyl compounds in heated cigarette-derived smoke extract (hCSE) and burned cigarette-derived smoke extract (bCSE) cause a reduction in endothelial nitric oxide synthase (eNOS) activity. Three types of heat-not-burn cigarettes (Ploom S, glo, and IQOS) and a combustion cigarette (hi-lite) were used to generate cigarette smoke at different heating or combustion temperatures [Ploom S (200°C), glo (240°C), IQOS (300–350°C), and hi-lite (770−870℃)]. The amounts of carbonyl compounds in hCSE/bCSE were assessed by measuring the carbonylation level of Ca2+-sensing receptor (CaSR) that promotes the phosphorylation of eNOS at Ser1177, which positively regulates eNOS activity. Although CaSR-mediated phosphorylation of eNOS were unaffected by hCSE from Ploom S and glo, hCSE/bCSE from IQOS and hi-lite reduced the eNOS phosphorylation. hCSE/bCSE from the cigarettes, except for that from Ploom S, facilitated carbonylation of CaSR with different potencies (rank order: glo < IQOS < hi-lite). The reduction of eNOS phosphorylation and the carbonylation of CaSR induced by hCSE/bCSE from IQOS and hi-lite were inhibited by treatment with mainstream smoke using a Carboxen-572 cartridge to scavenge carbonyl compounds. These results suggest that an increase in the heating or combustion temperature leads to an increase in the generation of carbonyl compounds, which cause endothelial dysfunction characterized by a reduction in eNOS activity., Japanese Pharmacological Society, Japanese - Molecular effects of cigarette smoke on airway epithelium cells
Higashi Tsunehito, Mai Yosuke, Mazaki Yuichi, Proceedings for Annual Meeting of The Japanese Pharmacological Society, 95, 1-P-076, 2022
Cigarette smoking is one of the risk factors in cardiovascular and respiratory diseases including atherosclerosis and chronic obstructive pulmonary disease (COPD). Although cigarette smoke mainstream consists of more than 4,500 chemical compounds, the compounds responsible for these diseases are still unknown. Cigarette smoke is divided in two phases: tar (particle) phase containing nicotine and gas phase. Airway epithelium cells are exposed both tar and gas phases. In this study, we have examined the effect of tar and gas phase of cigarette smoke on airway epithelium cells. The gas phase extract of cigarette smoke was prepared as previously described (Higashi et al., 2014, PLOS ONE 9: e107856). The tar phase extract of cigarette smoke was prepared by extracting tar phase on Cambridge filter with DMSO. Both tar and gas phases induced cell death in airway epithelial cells. The cell death induced by the gas phase was PKC-dependent, whereas the tar phase induced DNA double strand break and PKC-independent cell death. The pharmacological experiments revealed that the airway epithelium cell death by gas phase were ferroptosis. According to Yoshida et al., ferroptosis is involved in COPD pathogenesis (Yoshida et al., 2019, Nat Commun 10: 3145). Taken together, cigarette smoke gas phase might be a critical factor for cigarette smoking-induced COPD onset and development., Japanese Pharmacological Society, Japanese - Cigarette smoke gas phase induces ferroptosis via PKC in tracheal epithelial cells
Higashi Tsunehito, Mai Yosuke, Mazaki Yuichi, Proceedings for Annual Meeting of The Japanese Pharmacological Society, 96, 2-B-P-113, 2022
Cigarette smoking is one of the risk factors for respiratory diseases such as chronic obstructive pulmonary disease and emphysema. Cigarette smoke can be divided into two phases; tar (particle) phase including nicotine and gas phase. Although both phases of cigarette smoke have cytotoxic activity and affect respiratory system, the molecular mechanism for cytotoxicity has remained to be clarified. In this study, we have examined the effects of cigarette smoke extracts on tracheal epithelial cells and lung cells. Both tar and gas phases induced cell death. Ferrostatin-1 suppressed gas phase-induced cell death, whereas it had no effects on tar phase-induced cell death. Several unsaturated carbonyl compounds such as acrolein (ACR) and methyl vinyl ketone (MVK) are major cytotoxic compounds in the gas phase. Ferrostatin-1 also suppressed ACR- and MVK-induced cell death in tracheal epithelial cells. These results indicate that ACR and MVK in gas phase are critical factors for ferroptosis induction by cigarette smoke in the respiratory system. Since we have previously reported that ACR- and MVK-induced cell death is PKC-dependent in aorta smooth muscle cells, we have examined PKC inhibitors. The results suggest that novel and/or atypical PKCs involve in cigarette smoke-induced ferroptosis induction in tracheal epithelial cells., Japanese Pharmacological Society, Japanese - In vitroアッセイによる環境毒性因子の呼吸器系への影響の解明
東恒仁, 眞井洋輔, 真崎雄一, Program & Abstracts. Annual and International Meeting of the Japanese Association for Animal Cell Technology, 35th (CD-ROM), 2022 - Annexin A2 is involved in activation of AKT upon endothelin-1 stimulation in melanoma cells
Mazaki Yuichi, Higashi Tsunehito, Horinouchi Takahiro, Miwa Soichi, Proceedings for Annual Meeting of The Japanese Pharmacological Society, 94, 3-P1-30, 2021
Endothelin receptors (ETRs) is one of G protein coupled receptors, and consist of ET type A receptor (ETAR) and ET type B receptor (ETBR). The overexpression of endothelin (ET)-1 or ETRs is related to malignancy of human cancer, although ET-1 was originally identified as an endothelium-derived vasocontractile peptide. In cancer cells, ET-1 activates various signaling pathways, including mitogen-activated protein kinase, phosphatidylinositol 3-kinase, protein kinase C through ETRs, although the mechanisms by which ET-1 activates these signaling pathways remain uncertain. Previously, we found that ETRs interacted with annexin A2, which is overexpressed in various cancers, and annexin A2 silencing suppressed activation of ERK upon ET-1 stimulation in human umbilical vein cell line, EA.hy926 cells. Here we examine roles of annexin A2 in ET-1 signaling pathway of melanoma cells. ET-1 stimulation induces ATK activation in melanoma cells. On the other hand, annexin A2 silencing suppressed activation of AKT upon ET-1 stimulation. These results suggested that interaction of ETRs and annexin A2 play important roles in ET-1 signaling pathway of melanoma cells., Japanese Pharmacological Society, Japanese - Effects of gas phase extract of cigarette smokegenerated from heated tobacco and burned cigarette on human vascularendothelial cells
Horinouchi Takahiro, Mazaki Yuichi, Miwa Soichi, Proceedings for Annual Meeting of The Japanese Pharmacological Society, 94, 1-P2-10, 2021
The present study established a method to prepare heated tobacco-derivedcigarette smoke extract (hCSE) and burned cigarette-derived CSE (bCSE), andcompared their effects on human umbilical vein endothelial EA.hy926 cells. Threetypes of tobacco heating devices (Ploom S, glo, and IQOS) were used in order togenerate cigarette smoke at different heating temperatures. hCSE and bCSE wereprepared using the puffing regimen parameters of the CORESTA approach (55 mLpuff volume, 3 sec puff duration, and 1 puff every 30 sec). Tar phase (nicotineand tar) of cigarette smoke was removed by passing through a Cambridge glassfiber filter. The rank order of amounts of nicotine and tar trapped on theCambridge filter was Ploom S (6 ± 1 mg/91 puffs) < glo (16 ± 1 mg/91 puffs)< IQOS (56 ± 3 mg/91 puffs) < hi-lite (228 ± 2 mg/91 puffs). Crude hCSEand bCSE caused a decrease in mitochondrial metabolic activity and theexpression level of endothelial nitric oxide synthase, with the rank order ofpotency as follows: Ploom S < glo < IQOS < hi-lite. The reduction inmitochondrial metabolic activity was diminished by removing nicotine and tarfrom cigarette smoke with Cambridge filter. These results indicated that highercytotoxicity of cigarette smoke to vascular endothelial cells were correlatedwith higher heating/burning temperatures [Ploom S (200℃) < glo (240℃)Cytotoxic mechanisms and physiological effects of unsaturated carbonyl compounds
東恒仁, 真崎雄一, 環境ホルモン学会研究発表会プログラム・要旨集, 23rd (Web), 19, 19, 2021
日本内分泌撹乱物質学会, Japanese血管内皮細胞における加熱式たばこ主流煙水抽出物の作用解析
堀之内孝広, 真崎雄一, 三輪聡一, 日本薬理学雑誌, 156, Supplement, 2021The roles of Annexin A in ERK activation upon endothelin-1 stimulation
真崎雄一, 東恒仁, 堀之内孝広, 三輪聡一, 日本分子生物学会年会プログラム・要旨集(Web), 44th, 2021Annexin A2 is involved in activation of ERK upon endothelin-1 stimulation
Mazaki Yuichi, Higashi Tsunehito, Horinouchi Takahiro, Miwa Soichi, Proceedings for Annual Meeting of The Japanese Pharmacological Society, 93, 3-P-361, 2020
Endothelin receptors (ETRs) is one of G protein coupled receptors, and consist of ET type A receptor (ETAR) and ET type B receptor (ETBR). The overexpression of endothelin (ET)-1 or ETRs is related to malignancy of human cancer, although ET-1 was originally identified as an endothelium-derived vasocontractile peptide. In cancer cells, ET-1 activates various signaling pathways, including mitogen-activated protein kinase, phosphatidylinositol 3-kinase, protein kinase C through ETRs, although the mechanisms by which ET-1 activates these signaling pathways remain uncertain. Here, we found that ETRs interacted with annexin A2, which is overexpressed in various cancers. Annexin A2 bound to ETAR and ETBR. Upon ET-1 stimulation, serine phosphorylation of annexin A2 increased, while there is no change in tyrosine phosphorylation of annexin A2. Furthermore, we found that annexin A2 silencing suppressed activation of ERK upon ET-1 stimulation. These results suggested that interaction of ETRs and annexin A2 may play important roles in activation of extracellular signal-regulated kinase upon ET-1 stimulation., Japanese Pharmacological Society, JapaneseCa2+-sensing receptor-Gq/11 protein signaling pathway is involved in nitric oxide release from human vascular endothelial cells
Horinouchi Takahiro, Mazaki Yuichi, Miwa Soichi, Proceedings for Annual Meeting of The Japanese Pharmacological Society, 93, 1-LBS-11, 2020
Ca2+-sensing receptor (CaSR) belongs to family C of G protein-coupled receptors and is activated by the endogenous agonists such as Ca2+. Stimulation of CaSR expressed in vascular endothelial cells through the increase in extracellular Ca2+ concentration ([Ca2+]o) is reported to induce vasorelaxation via the production of nitric oxide (NO). The purpose of the present study is to characterize the CaSR-mediated NO production in human vascular endothelial cells. In human endothelial EA.hy926 cells, the increase in [Ca2+]o from 0.2 to 2 mM induced a concentration-dependent increase in intracellular Ca2+ concentration, which was significantly inhibited by NPS 2143 (a CaSR antagonist) and YM-254890 (a Gq/11 protein inhibitor). Stimulation with 2 mM Ca2+ for 4 h elicited an increase in the phosphorylation level of eNOS at Ser1177, which was significantly depressed by NPS 2143, YM-254890, and removal of Ca2+ from the medium. Ca2+ (2 mM) induced an increase in NO production, which was inhibited by NPS 2143, YM-254890, removal of Ca2+ from the medium, and L-NAME (a competitive eNOS inhibitor). These results provide evidence that activation of CaSR with extracellular Ca2+ facilitates NO release from human vascular endothelial cells via a Gq/11 protein-eNOS-dependent pathway., Japanese Pharmacological Society, JapaneseMolecular mechanisms for cigarette smoke tar phase-induced cell death
Higashi Tsunehito, Mazaki Yuichi, Proceedings for Annual Meeting of The Japanese Pharmacological Society, 93, 3-P-379, 2020
Cigarette smoke is divided in tar phase containing nicotine and gas phase. The gas phase of cigarette smoke is prepared by passing cigarette smoke through Cambridge filter. The tar phase is extracted from Cambridge filter by 2-propanol. We have previously elucidated that the gas phase induce cell death by intracellular Ca2+- and protein kinase C (PKC)-dependent manner, and identified acrolein and methyl vinyl ketone as the major cytotoxic compounds in the gas phase (Mai et al., 2012; Noya et al., 2013; Higashi et al., 2014). In this study, we examined molecular mechanism(s) for cigarette smoke tar phase-induced cell death in lung cancer cells. Lung adenocarcinoma, small cell carcinoma, and non-small cell carcinoma cell lines are all sensitive to cigarette smoke tar phase. Tar phase-induced cell death is intracellular Ca2+- and PKC-independent, whereas intracellular Ca2+ chelator and PKC inhibitor effectively suppressed gas phase-induced cell death. These results indicate that the molecular mechanisms for cell death induction by cigarette tar phase is different from that of cigarette smoke gas phase., Japanese Pharmacological Society, Japanese不飽和カルボニル化合物による細胞死に関与するPKCアイソフォームの同定
東恒仁, 真崎雄一, 日本分子生物学会年会プログラム・要旨集(Web), 43rd, 2020血管内皮細胞におけるカルシウム感受性受容体を介した一酸化窒素産生機序
堀之内孝広, 真崎雄一, 寺田晃士, 三輪聡一, 日本薬理学雑誌, 155, Supplement, 2020好中球様細胞に分化させたHL-60細胞のケモタキシスにおける小胞体とミトコンドリアの接触
真崎雄一, 高田真吾, 小林純子, 前川聡, 小野寺康仁, 佐邊壽孝, 日本分子生物学会年会プログラム・要旨集(Web), 43rd, 2020GRP78 promotes ERK activation through endothelin type B receptor
Mazaki Yuichi, Higashi Tsunehito, Onodera Yasuhito, Nam Jin-Min, Hashimoto Ari, Hashimoto Shigeru, Horinouchi Takahiro, Miwa Soichi, Proceedings for Annual Meeting of The Japanese Pharmacological Society, 92, 1-P-110, 2019
Endothelin (ET)-1 is involved in various diseases, including cancer, hypertension, atherosclerosis, diabetes, and fibrotic diseases, although ET-1 is originally identified as endothelium-derived vasocontractile peptide. ET receptors belong to the class A of G protein-coupled receptor, and consist of ET type A receptor (ETAR) and ET type B receptor (ETBR). ETAR and ETBR generally exhibit the opposite responses, although many exceptions exist. Here, we attempted to identify ETAR or ETBR specific binding proteins to understand difference of ETAR- and ETBR-mediated responses upon ET-1 stimulation. We found that GRP78 exhibited a stronger binding affinity toward ETBR than ETAR. Overexpression of GRP78 promotes ETBR-mediated ERK activation. In addition, the silencing of GRP78 suppressed ETBR-mediated ERK activation. On the other hand, ETBR can localize GRP78 to cell periphery. Our results suggest that interaction of ETBR with GRP78 affects the ERK activation and GRP78 localization., Japanese Pharmacological Society, JapaneseElucidation of molecular mechanism for detoxification of cigarette smoke gas phase by antioxidants
Higashi Tsunehito, Elmeligy Enas, Mai Yosuke, Noya Yoichi, Kuge Yuji, Mazaki Yuichi, Miwa Soichi, Proceedings for Annual Meeting of The Japanese Pharmacological Society, 92, 1-P-121, 2019
Unsaturated carbonyl compounds such as acrolein (ACR) and methyl vinyl ketone (MVK) are major cytotoxic factors in the gas phase extract of cigarette smoke. ACR and MVK induce cell membrane damage and cell death through protein kinase C and NADPH oxidases. We have previously reported that several antioxidants such as reduced glutathione (GSH) and N-acetylcysteine (NAC) can suppress ACR- and MVK-induced cell membrane damage and cell death, although the molecular mechanism has remained to be clarified. In this study, we have elucidated the mechanism for suppression of ACR- and MVK-induced cell injury by antioxidants. The molecules with thiol group such as GSH, NAC, and cysteine effectively suppressed cell membrane damage and cell death induced by cigarette smoke gas phase, ACR, and MVK. The results of HPLC and MS showed that GSH and NAC directly reacted with ACR and MVK. Cysteine and cysteine derivatives suppressed ACR-induced protein carbonylation. Current results suggest that GSH, NAC, and cysteine directly reacted with ACR and MVK, and suppressed unsaturated carbonyl compounds-induced cell damage by preventing protein carbonylation., Japanese Pharmacological Society, JapaneseInvolvement of Ca2+-sensing receptor in activation of nitric oxide synthase of human vascular endothelial cells.
Horinouchi Takahiro, Mazaki Yuichi, Miwa Soichi, Proceedings for Annual Meeting of The Japanese Pharmacological Society, 92, 1-O-01, 2019
Ca2+-sensing receptor (CaSR) is a seven-transmembrane G protein-coupled receptor (GPCR), and is activated by an increase in extracellular Ca2+ concentration. In vascular endothelial cells, stimulation of CaSR induces nitric oxide (NO) release via activation of endothelial nitric oxide synthase (eNOS) and membrane hyperpolarization via activation of intermediate Ca2+-activated K+ channels, contributing to vasodilation. In the present study, we have pharmacologically characterized eNOS activation in response to stimulation of CaSR in human endothelial EA.hy926 cells. In 2 mM Ca2+-containing Krebs-HEPES solution, the phosphorylation level of eNOS at serine 1177 was markedly reduced by NPS 2143 (a CaSR antagonist) and YM-254890 (a Gq/11 protein inhibitor). In organ bath study with endothelium-removed ring preparations of rat thoracic aorta, addition of EA.hy926 cell suspension produced relaxation of the rings precontracted with phenylephrine. The endothelium-dependent relaxant response was inhibited by pretreatment of EA.hy926 cells with NPS 2143, YM-254890, and L-NAME (an eNOS inhibitor). These results suggest that stimulation of CaSR expressed in endothelial cells with extracellular Ca2+ induces NO-mediated vasorelaxation via Gq/11-protein-dependent activation of eNOS., Japanese Pharmacological Society, Japaneseシステイン誘導体による煙中の不飽和カルボニル化合物の解毒メカニズムの解明
東恒仁, ELMELIGY Enas, 眞井洋輔, 野矢洋一, 真崎雄一, 久下裕司, 三輪聡一, 日本生物工学会大会講演要旨集, 71st, 134, 134, 2019
(公社)日本生物工学会, Japanese血管内皮細胞におけるカルシウム感受性受容体を介したNO産生に対するタバコ煙ガス相水抽出物及びアクロレインの抑制作用
堀之内孝広, 真崎雄一, 三輪聡一, 日本薬理学雑誌, 153, Supplement, 2019MFN2は好中球様細胞に分化させたHL-60細胞のケモタキシスに関与する
真崎雄一, 高田真吾, 小林純子, 前川聡, 東恒仁, 小野寺康仁, 佐邊壽孝, 日本分子生物学会年会プログラム・要旨集(Web), 42nd, 2019【心脈管作動物質研究の新潮流】エンドセリンによる骨格筋でのインスリン抵抗性の発症機序
堀之内 孝広, 真崎 雄一, 寺田 晃士, 三輪 聡一, 日本薬理学雑誌, 151, 4, 140, 147, Apr. 2018
(公社)日本薬理学会, Japanese不飽和カルボニル化合物が血管構成細胞に及ぼす影響の解明
東恒仁, 眞井洋輔, 真崎雄一, 日本生物工学会大会講演要旨集, 70th, 134, 134, 2018
(公社)日本生物工学会, Japanese不飽和カルボニル化合物はプロテインキナーゼC依存的に心血管系細胞の細胞死を誘導する
東恒仁, 眞井洋輔, 真崎雄一, 日本分子生物学会年会プログラム・要旨集(Web), 41st, 2018エンドセリンB受容体はGRP78と相互作用する
真崎雄一, 東恒仁, 堀之内孝広, 橋本あり, 橋本茂, 南ジンミン, 小野寺康仁, 日本分子生物学会年会プログラム・要旨集(Web), 41st, 2018血管内皮細胞におけるアクロレインの作用解析
堀之内孝広, 三輪聡一, 三輪聡一, 真崎雄一, 東恒仁, 日本薬理学会北部会プログラム・抄録集, 68th, 2017不飽和カルボニル化合物による細胞死誘導の分子機構の解明
東恒仁, 眞井洋輔, 真崎雄一, 日本生化学会大会(Web), 90th, [1P, 0456], 2017
生命科学系学会合同年次大会運営事務局, Japanese好中球のケモタキシスにおいて,ARF1の活性化は,ARF1‐RAC1の相互制御回路を開始する
真崎雄一, 小野寺康仁, 東恒仁, 堀之内孝広, 及川司, 佐邊壽孝, 日本細胞生物学会大会(Web), 69th, ROMBUNNO.T8‐11(P1‐077) (WEB ONLY), 63, 2017
(一社)日本細胞生物学会, JapanesePhos-tag電気泳動法の応用 Phos-tag biotinを用いた受容体作動性TRPC6チャネルのPKAリン酸化部分の同定
堀之内 孝広, 寺田 晃士, 東 恒仁, 真崎 雄一, 三輪 聡一, 電気泳動, 60, Suppl., s19, s19, Aug. 2016
日本電気泳動学会, Japaneseエンドセリン受容体シグナルを標的にした肺高血圧症治療の新展開
堀之内 孝広, 堀口 美香, 真崎 雄一, 東 恒仁, 原田 拓弥, 三輪 聡一, 呼吸と循環, 64, 5, S9, S10, May 2016
(株)医学書院, Japanese肺高血圧症治療薬の最近の進歩:エンドセリンシステムからみた作用機序
堀之内孝広, 真崎雄一, 寺田晃士, 東恒仁, 三輪聡一, 日本薬理学雑誌, 148, 5, 231, 238, 2016
(公社)日本薬理学会, JapanesePhos-tag biotinを用いた受容体作動性TRPC6チャネルのPKAリン酸化部位の同定
堀之内孝広, 寺田晃士, 東恒仁, 真崎雄一, 三輪聡一, 電気泳動(Web), 60, Suppl, 2016Unsaturated carbonyl compounds in the gas phase of cigarette smoke induces cell death through intracellular Ca2+-dependent PKC activation.
T. Higashi, Y. Mai, Y. Mazaki, T. Horinouchi, S. Miwa, MOLECULAR BIOLOGY OF THE CELL, 27, 2016
English, Summary international conference肺血管リモデリングの発症機序に迫る エンドセリン受容体シグナルからの視点
堀之内 孝広, 東 恒仁, 真崎 雄一, 三輪 聡一, 東邦医学会雑誌, 62, 3, 197, 199, Sep. 2015
東邦大学医学会, JapaneseNew insights regarding the pathogenic mechanisms underlying pulmonary vascular remodeling from endothelin receptor signaling
堀之内 孝広, 東 恒仁, 真崎 雄一, Toho journal of medicine, 1, 3, 197, 199, Sep. 2015
The Medical Society of Toho University, Japanese細胞骨格・細胞運動・細胞移動 GBF1のSec7ドメインのHomology Downstreamは、化学走化性とスーパーオキシド産生に重要な役割を果たすArf活性を持つGPCRシグナル伝達とリンクするホスファチジルイノシトールリン酸と結合する(Cytoskeleton/Cell motility/Cell migration The Homology Downstream of Sec7 domain of GBF1 binds to phosphatidyl inositol phosphates to
真崎 雄一, 佐邊 壽孝, 日本細胞生物学会大会講演要旨集, 63回, 116, 116, May 2011
(一社)日本細胞生物学会, English好中球においてゴルジ体に局在するArfGEFのPI3Kγによる局在変化と活性化は、GPCR刺激と細胞運動を結びつけている(Translocation and activation of a Golgi-localizing ArfGEF via PI3Kγ links GPCR stimulation with directional migration in neutrophils)
真崎 雄一, 佐邊 壽孝, 日本細胞生物学会大会講演要旨集, 62回, 138, 138, May 2010
(一社)日本細胞生物学会, English好中球のケモタキシスにおけるGBF1の役割(Roles of GBF1 in neutrophil chemotaxis)
真崎 雄一, 佐邊 壽孝, 日本細胞生物学会大会講演要旨集, 61回, 148, 148, May 2009
(一社)日本細胞生物学会, English癌浸潤転移における細胞運動のメカニズム 血管新生と癌浸潤に共通なシグナル経路(Molecular mechanisms of cell migration in cancer invasion and metastasis Common usage of an Arf6-GEP100 signaling pathway in angiogenesis and tumor invasion)
橋本 あり, 橋本 茂, 小川 栄治, 廣瀬 まゆみ, 高島 成二, 森重 真毅, 毛受 暁史, 南 ジンミン, 真崎 雄一, 北風 政史, 渋谷 正史, 佐邊 壽孝, 日本細胞生物学会大会講演要旨集, 60回, 95, 95, Jun. 2008
(一社)日本細胞生物学会, EnglishFbx8によるユビキチン化を介するArf6の抑制的制御と上皮組織形態形成との関連の可能性について(Fbx8 makes Arf6 refractory to function via ubiquitination: implication in epithelial tissue organization)
矢野 元, 小野寺 康仁, 鳥井 郁子, 真崎 雄一, 橋本 茂, 辻村 亨, 佐邊 壽孝, 日本生化学会大会・日本分子生物学会年会合同大会講演要旨集, 80回・30回, 1T21, 3, Nov. 2007
(公社)日本生化学会, EnglishArf6をユビキチン化するE3リガーゼFbx8のがんにおける発現不全(Loss of Fbx8, a component of E3 ligase mediating Arf6 ubiquitination, in different human tumors)
矢野 元, 真崎 雄一, 橋本 茂, 辻村 亨, 佐邊 壽孝, 日本癌学会総会記事, 66回, 142, 143, Aug. 2007
日本癌学会, English[Directional sensing and superoxide production in neutrophils].
Yuichi Mazaki, Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme, 51, 6 Suppl, 727, 32, May 2006, [Peer-reviewed], [Domestic magazines]
共立出版, Japanese浸潤性乳癌細胞におけるAMAP1の発現と機能
小野寺 康仁, 橋本 茂, 真崎 雄一, 橋本 あり, 森重 真毅, 松浦 成昭, 佐邊 壽孝, 日本癌学会総会記事, 63回, 267, 267, Sep. 2004
日本癌学会, JapaneseRoles played by a subset of integrin-signaling molecules in cadherin-based cell-cell adhesion
Hajime Yano, Yuichi Mazaki, Kazuo Kurokawa, Steven Hanks, Michiyuki Matsuda, Hisataka Sabe, CELL STRUCTURE AND FUNCTION, 29, 32, 32, May 2004
English, Summary international conferenceカドヘリン接着形成におけるインテグリンシグナル分子群の役割
矢野 元, 真崎 雄一, 三浦 浩一, 佐邊 壽孝, 日本癌学会総会記事, 62回, 40, 40, Aug. 2003
日本癌学会, Japanese細胞外マトリックス系による細胞増殖と機能の制御 運動中の細胞における,パキシリンのチロシン31及び118のリン酸化により制御されるRhoA活性抑制の局在化機構
坪内 朝子, 坂倉 純子, 八木 良平, 真崎 雄一, Schaefer Erik, 矢野 元, 佐邊 壽孝, 日本発生生物学会大会講演要旨集, 35回, 98, 98, May 2002
日本発生生物学会, Japaneseダイナミックな細胞骨格制御と細胞機能 パキシリン結合性ARFGAP蛋白質のアクチン細胞骨格制御における役割
真崎 雄一, 佐邊 壽孝, 日本細胞生物学会大会講演要旨集, 54回, 3, 3, May 2001
(一社)日本細胞生物学会, JapaneseInteraction of paxillin with p21-activated kinase (PAK)
S Hashimoto, A Tsubouchi, Y Mazaki, H Sabe, MOLECULAR BIOLOGY OF THE CELL, 11, 172A, 173A, Dec. 2000
English, Summary international conference細胞接着と細胞骨格の制御と細胞形態形成 細胞骨格制御におけるパキシリンと低分子量G蛋白質群との機能連関
佐邊 壽孝, 橋本 茂, 近藤 明子, 坪内 朝子, 内田 浩, 中村 邦明, 矢野 元, 真崎 雄一, 生化学, 72, 8, 593, 593, Aug. 2000
(公社)日本生化学会, JapaneseARF GAP活性を有するPagはゴルジ構造とパキシリンの細胞内局在制御に関与する
真崎 雄一, 矢野 元, 大川 克也, 岩松 明彦, 佐邊 壽孝, 日本癌学会総会記事, 58回, 184, 184, Aug. 1999
日本癌学会, JapaneseARF GAP活性を有するパキシリン結合性新規タンパク質
近藤 明子, 橋本 茂, 真崎 雄一, 佐邊 壽孝, 日本癌学会総会記事, 58回, 184, 184, Aug. 1999
日本癌学会, JapaneseARF GAP活性を示すパキシリン結合タンパク質PAGsの計算機データベースでの検索
内田浩, 真崎雄一, 近藤明子, 佐辺寿孝, 日本分子生物学会年会プログラム・講演要旨集, 22nd, 1999パキシリン結合タンパク質PAG1はARF1 GAP活性を示し,ゴルジ構造とパキシリンの細胞内局在制御に関与する
真崎雄一, 矢野元, 八木良平, 近藤明子, 大川克也, 岩松明彦, 佐辺寿孝, 日本分子生物学会年会プログラム・講演要旨集, 22nd, 1999ARF6 GAP活性を示すパキシリン結合性タンパク質PAG3は細胞運動性を制御する
近藤明子, 橋本茂, 矢野元, 永山国昭, 真崎雄一, 佐辺寿孝, 日本分子生物学会年会プログラム・講演要旨集, 22nd, 1999Paxillin function during epitherial-mesenchymal transdifferentiation
UCHIDA Hiroshi, YANO Hajime, MAZAKI Yuichi, HASHIMOTO Shigeru, SABE Hisataka, 日本分子生物学会年会プログラム・講演要旨集, 21, 0, 521, 521, 01 Dec. 1998
JapaneseA novel protein that anchors a focal adhesion protein paxillin to the perinuclear regions
MAZAKI Yuichi, YANO Hajime, OKAWA Katsuya, HASHIMOTO Shigeru, IWAMATSU Akihiro, SABE Hisatake, 日本分子生物学会年会プログラム・講演要旨集, 21, 0, 522, 522, 01 Dec. 1998
JapaneseAnalysis of the novel paxillin binding protein
KONDO Akiko, HASHIMOTO Shigeru, MAZAKI Yuichi, NAGAYAMA Kuniaki, SABE Hisataka, 日本分子生物学会年会プログラム・講演要旨集, 21, 0, 530, 530, 01 Dec. 1998
JapaneseEpithelial cell survival signals
HASHIMOTO Shigeru, MAZAKI Yuichi, UCHIDA Hirosi, SABE Hisataka, 日本分子生物学会年会プログラム・講演要旨集, 21, 0, 544, 544, 01 Dec. 1998
Japanese膜裏打ち分子による細胞内情報伝達 上皮間充織形質転換と細胞生存性・運動性の制御 インテグリン裏打ち蛋白質パキシリン(Paxillin)を中心として
佐邊 壽孝, 真崎 雄一, 内田 浩, 橋本 茂, 生化学, 70, 8, 703, 703, Aug. 1998
(公社)日本生化学会, Japanese接着斑タンパク質パキシリン(Paxillin)のゴルジ装置への局在について
真崎 雄一, 大川 克也, 内田 浩, 橋本 茂, 岩松 明彦, 佐邊 壽孝, 日本癌学会総会記事, 57回, 146, 146, Aug. 1998
日本癌学会, Japaneseヒト癌におけるpaxillin isoformの発現の解析
内田 浩, 真崎 雄一, 橋本 茂, 佐邊 壽孝, 日本癌学会総会記事, 57回, 451, 451, Aug. 1998
日本癌学会, Japanese上皮系細胞のインテグリンを介する生存性維持機構に基付いた細胞癌化機構の解析
橋本 茂, 真崎 雄一, 内田 浩, 佐邊 壽孝, 日本癌学会総会記事, 57回, 450, 450, Aug. 1998
日本癌学会, Japanese細胞接着とシグナル伝達 インテグリンを介した細胞基質間接着におけるシグナル伝達
真崎 雄一, 佐邊 壽孝, 組織培養工学, 23, 6, 218, 222, May 1997
(株)ニュー・サイエンス社, JapaneseAnalysis of paxillin isoforms in mice.
真崎雄一, 橋本茂, 佐辺寿孝, 日本分子生物学会年会プログラム・講演要旨集, 20th, 1997Analysis of a bHLHzip gene, mi, regulating differentiation of pigmented epithelial cells.
餅井真, 真崎雄一, 江口吾朗, 日本発生生物学会大会発表要旨集, 29th, 1996The role of integrins in transdifferentiation of chicken retinal pigmented epithelial cell.
真崎雄一, 餅井真, 江口吾朗, 日本発生生物学会大会発表要旨集, 28th, 1995Expression of integrins in chicken retinal pigmented epithelial cell.
真崎雄一, 餅井真, 江口吾朗, 日本分子生物学会年会プログラム・講演要旨集, 17th, 1994Affiliated academic society
Research Themes
- 感染時にみられる好中球のエネルギー産生の場の変更機構の解明
科学研究費助成事業
01 Apr. 2021 - 31 Mar. 2024
真崎 雄一
好中球は、感染初期に働く重要な免疫細胞の一つである。通常、好中球は、活性酸素種の産生を抑えるために、解糖系を用いてATPを産生している。しかし、体内に病原体が侵入すると、大量のATPを必要とするため、ミトコンドリアを使ってATPを産生するようになる。これまでに、申請者は、ジメチルスルホキシドによってHL-60細胞を好中球細胞様へ分化させた細胞（dHL-60細胞）を細菌性ペプチドN-formyl-Met-Leu-Phe（fMLP）で刺激すると、極めて短時間にミトコンドリアの形態が変化し、酸化的リン酸化の量が増加すること。ミトコンドリア融合関連タンパク質Mitofusin 2（MFN2）の発現を抑えと、ミトコンドリアの形態変化と酸化的リン酸化の量が減少すること。また、fMLPの刺激による遊走能（ケモタキシス）も減少することを明らかにし報告した。さらに、最近、MFN2結合タンパク質の発現を抑えると、ミトコンドリアの形態変化とケモタキシスが抑えられるという結果を得ている。そこで、本研究では、MFN2とMFN2結合タンパク質の関係を中心に、感染に伴って起こる好中球のエネルギー産生の場（ATP産生の場）の変更機構を明らかにすることを目的とし研究を開始した。
今年度は、これまでdHL-60細胞で見出していた現象が、好中球でも見られるのか検証するために、MFN2結合タンパク質のノックアウトマウスの好中球を用い、このタンパク質が好中球のケモタキシスに関与しているのか調べた。その結果、dHL-60細胞で得ていた結果と同様、MFN2結合タンパク質のノックアウトマウスの好中球でも、正常マウスの好中球と比べ、ケモタキシスが減少していることが明らかになった。
日本学術振興会, 基盤研究(C), 北海道大学, 21K08487 - Elucidation of the mechanisms of neutrophil mitochondrial fusion in infection
Grants-in-Aid for Scientific Research
01 Apr. 2018 - 31 Mar. 2021
Mazaki Yuichi
Neutrophils rapidly migrate to infection sites after the recognition of invaders. Previously, we found that mitochondrial morphology changes to a tubular form after fMLP stimulation. In addition, mitochondria oxidative phosphorylation (OXPHOS) activity significantly increased after fMLP stimulation. In this study, we examined mechanisms of mitochondrial morphology changes after fMLP stimulation. We found that the silencing of mitochondrial fusion protein Mitofusin 2 (MFN2) suppresses mitochondrial morphological changes. Furthermore, MFN2 silencing suppressed OXPHOS activation and chemotaxis upon fMLP stimulation.
Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (C), Hokkaido University, 18K08423 - Roles of ARFs in superoxide production and chemotaxis of neutrophil
Grants-in-Aid for Scientific Research
01 Apr. 2013 - 31 Mar. 2016
MAZAKI Yuichi, SABE Hisataka
Neutrophils are important in innate immunity and in the initiating an acute response to infection. During such a response, neutrophils migrate towards invaders and produce antimicrobial agents, including many reactive oxygen species. Previously, we reported that ARF regulatory factors, which are membrane trafficking protein, are involved in superoxide production and chemotaxis of neutrophils. In this study, we examined roles of ARFs in superoxide production and chemotaxis of neutrophil. Our results suggest that ARF1 is involved in subcellular localization of Rac1 during chemotaxis.
Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (C), 25440051 - Roles of GBF1 in cell migration of neutrophil
Grants-in-Aid for Scientific Research
2009 - 2011
MAZAKI Yuichi
It is known that neutrophils rapidly migrate into the infection sites of invading pathogens, although the molecular mechanism remains largely unknown. In this study, to resolve the molecular mechanism of cell migration in neutrophil, we examined the activation mechanism and roles of GBF1, intercellular trafficking associated protein, in cell migration. We found that GBF1 is activated by PI(3, 4, 5) P_3, and that GBF1 is involved in subcellular localization of ARF1 and GIT2 during cell migration.
Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (C), Kumamoto University, 21570201 - Roles of ARF in polarity formation during cell migration
Grants-in-Aid for Scientific Research
2007 - 2008
MAZAKI Yuichi
細胞運動は、我々の身体の至る所で見られ、それらは厳密にコントロールされており、一旦、異常が起こると、我々の身体に重篤な障害をもたらす。このように、細胞運動は、重要な生命現象であり、その仕組みが明らかになれば、病気の治療や解明といった様々な恩恵を我々は得ることができると期待される。近年の研究によって、細胞の移動の仕組みは明らかになりつつあるものの、移動の前段階である極性形成については、不明な点が多い。本研究では、この点に注目し、免疫細胞の一つである好中球の細胞に関与する分子を調べた。その結果、細胞内輸送に関与するGBF1が、好中球の細胞運動に極めて重要な役割を果たしていることが明らかとなった。
Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (C), Kumamoto University, 19570191 - Roles played by Arf6 in cell migration and invasion
Grants-in-Aid for Scientific Research
2004 - 2005
SABE Hisataka, HASHIMOTO Shigeru, MIURA Koichi, HASHIMOTO Ari
Cell migration is a multifactorial process in which a number of distinct events occur simultaneously. The major purpose of this study is to understand basic molecules and mechanisms coordinately regulating cell migration and invasion.
Arf6 plays essential roles in recycling of plasma membrane component, as well as both membrane and cytoskeletal remodeling at cell peripheries. We have previously identified several proteins bearing ArfGAP domains as binding proteins to paxillin, an integrin signaling adaptor/scaffolding protein. These ArfGAPs include AMAP1 and AMAP2. We have also shown that AMAP2 has a role in recruiting paxillin to sites of focal adhesions in epithelial cells. We have further demonstrated that AMAP1 is crucial for invasive activities of different breast cancer cells, in which AMAP1 functions by forming a trimeric protein complex with paxillin and cortactin. siRNA-mediated knockdown of AMAP1 effectively block invasive and metastatic activities of breast cancer cells.
Then, there was an enigma why GAP protein like AMAP1 can act as a necessary factor for tumor invasion, in spite of the simple idea that it may rather act to inhibit the invasion. Indeed, biochemical assays have shown a lack of efficient GAPing activity of AMAP1 against Arf6. We thus tested a mode of interaction between AMAP1 and Arf6, and found that AMAP1 via its ArfGAP domain binds to GTP-Arf6 stably, even in the presence of divalent cations. Binding of AMAP1 to other GTP bound Arfs, like Arf1 and Arf5, was negligible. We also conducted several cell biological assays, and concluded that AMAP1, and also AMAP2, act as effectors for GTP-Arf6. Both paxillin and cortactin are known to be essential for invadopodia formation, that are sites of tumor cell invasion into basement membranes. We showed that by binding to GTP-Arf6, AMAP1 has a role in recruiting paxillin and cortactin to invadopodia in breast cancer cells. Next we addressed to identify a GEF that is responsible for Arf6 activation in migration and invasion. We have succeeded in this identification, and also identified signaling pathways as to how this Arf6GEF becomes activated by extracellular stimuli. Besides these, we also showed that Arf6 can be ubiquitinated. We identified a E3 ligase involved in this ubiquitination, and shown that this is a non-canonical ubiquitination and that loss of the E3 ligase expression may contribute to invasive phenotypes tumor cells.
Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (B), OSAKA BIOSCIENCE INSTITUTE, 16370090 - Role of paxillin-associatcd ARFGAPs in cell migration.
Grants-in-Aid for Scientific Research
2000 - 2001
SABE Hisataka, YAGI Ryohei, YANO Hajime, HASIMOTO Sigeru
ARF6 regulates endosomal recycling. We have shown that PAG3/Papα/KIAA0400 acts as a GTPase-activation protein (GAP) specific for ARF6.We study here molecular mechanims how PAG3 is involved in endosomal recycling to be an ARF6GAP. We found that PAG3, via its proline-rich region, binds to the src homology 3 (SH3) domain of several components of the endocytic machinery, and analysed its interaction with amphiphysin IIa. PAG3 existed at ARF6(Q67L)-positive membrane ruffles colocalized with amphyphysin Ha, but the majority exists at intracellular tubulovesicular structure. Overexpression of the amphiphysin ha SH3 domain is known to block endocytosis. Likewise, overexpression of the proline-rich region of PAG3 blocked both clathrin-dependent and independent endocytosis, while mutations of amino acids essential for the binding abolished such blockage. The SH3 domain of amphiphysin IIa. also binds to dynamin, a mechano-enzyme essential for the late step of endocytosis. We found that PAG3 exhibits almost one order of magnitude higher affinity than that of dynamin towards amphiphysin ha. We also demonstrated that PAG3 can be phosphorylated by a protein tyrosine kinase, Pyk2, but not by its close relative Fak ; and this phosphorylation inhibits the association with amphiphysin ha. With further results, we propose that PAG3 recruits amphiphysin ha to the plasma membrane, probably through interaction with the activity of GTP-bound Arf6 ; and external stimuli triggering endocytosis evoke tyrosine phosphorylation of PAG3, the phosphorylated PAG3 then releases amphiphysin ha to associate with components of endocytic machinery such as dynamin.
Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (B), OSAKA BIOSCIENCE INSTITUTE(OBI), 12480219 - マクロファージの泡沫化に関わる分子の検索および解析
科学研究費助成事業
2000 - 2001
真崎 雄一, 近藤 明子
粥状硬化は、高血圧を含む多くの循環器疾患を引き起こす原因の一つである。粥状硬化は、血管内皮に浸潤したマクロファージが、特に、酸化LDLを取り込むことによって泡沫細胞化し、その後、内膜に蓄積することが原因であると考えられている。酸化LDLは、スカベンジャーレセプター等を介してマクロファージ内へ積極的に取り込まれるとともに、マクロファージの遊走能に対して促進活性を持つことが報告されている。一方、インテグリン裏打ちタンパク質であるパキシリン及びその結合タンパク質が、単球細胞及びマクロファージの浸潤、運動過程、さらに貪食過程にも関与することが明らかとなってきた。本研究では、パキシリン結合タンパク質に共通するドメインに注目し、遺伝子データベースによる検索、構造分類を行う。さらに、それら遺伝子のマクロファージの運動及び貪食過程における関与、酸化LDLを貪食させた際の発現パターンを調べることで、マクロファージの泡沫化に関与する遺伝子の探査解明を行い、粥状硬化の予防、診断、治療に貢献することを目標としている。
本年度は、パキシリン結合タンパク質に共通するドメインであるARFGAPドメインに注目し、検索の結果、9つのタンパク質がこれに該当することが明らかとなった。これらは、アンキリンモチーフを持つもの(7つ)と持たないもの(2つ)の2つの群に分類することができた。これら全てのタンパク質を293T細胞を使い発現させ、パキシリンとの結合を調べた。その結果、アンキリンモチーフを持つものでは、発現しなかった1つ除く、残り6つ全てのタンパク質がパキシリンに結合したのに対し、アンキリンモチーフを持たないものでは、いずれもパキシリンに対して結合を示さなかった。さらに、アンキリンモチーフを持つものうち、これまで我々が解析を進めてきたPAG1とPAG3について、マクロファージの貪食過程における関与を調べた結果、PAG1が貪食過程においてほとんど影響を示さなかったのに対し、PAG3は、貪食過程において深く関与することが明らかとなった。
日本学術振興会, 特定領域研究(C), (財)大阪バイオサイエンス研究所, 12204109 - インテグリンを介する細胞接着シグナル制御と癌化細胞の生存性・侵潤能の解析
科学研究費助成事業
1999 - 2001
佐邊 壽孝, 真崎 雄一, 矢野 元, 橋本 茂, MARIUS Sudol, DONALD E. Ingber, JOAN S. Brugge
細胞が運動する際には、細胞の前方部にインテグリン接着点が新たに形成される。インテグリンは多くの種類の蛋白質をその細胞質領域に集積させることにより機能する。我々は、「このような裏打ち蛋白質群を集積させる過程が単に細胞質における自由拡散過程なのか、それとも、何か能動的な機序が存在するのか」との設問を立て、解析を開始した。
それまで機能解析をしてきたパキシリンというインテグリン裏打ち蛋白質に関して上記のような解析をしたところ、このものは、核周辺領域に細胞質プールが存在し、運動中に細胞前方に形成されるラミニポデアにパキシリンを集積させる過程は、細胞質での自由拡散ではなく、何らかの能動的な機序が存在することを強く示唆する結果を得た。そこで、パキシリンをプローブとしパキシリンの細胞内動態を説明できるような蛋白質を検索したところ、小胞/膜/蛋白質の輸送に関与する低分子性GTP結合蛋白質であるARF蛋白質に対するGAP(GTPase-activating protein)活性を持つ一連の興味深い一連の蛋白質が得られ、これらの蛋白質をPAGs(Paxillin-associated ARFGAP proteins)と命名した。現在、5種類のcDNAが得られており、その内、2種に関してほぼ初期段階の解析が終了し、論文に纏めている。ARFは哺乳類では6種のアイソフォームが存在するが、解析の終わった2種のPAG蛋白質はin vivoでそれぞれ異なったARFアイソフォームに対するGAP活性を示すこと、それらはパキシリンの細胞内での局在とダイナミックスの制御に互いに異なった機序により関与していること、さらには、細胞運動の制御にも関与していることを明らかにしている。特に、パキシリン結合性蛋白質として単離したこのようなPAG蛋白質が全てARFGAP活性を持っていることは、パキシリンのフォーカルコンタクトへの集積過程は、その過程自身に対して何らかの負のフィードバック制御機構を持っていることを示唆している。また、単球の成熟過程において発現誘導されるPAGが存在し、それは、ヒト動脈硬化病変における泡沫細胞に高レベル発現していることも観察している。
日本学術振興会, 特定領域研究(A), (財)大阪バイオサイエンス研究所, 11137310 - インテグリンを介する細胞接着シグナル制御と癌化細胞の生存性・浸潤能の解析
科学研究費助成事業
2000 - 2000
橋本 茂, 中村 邦明, 矢野 元, 真崎 雄一, 八木 良平
細胞が運動する際、前方にfilopodiaとlamellipodiaが形成され、focal complexの形成を経て、細胞体の前方への移動に伴いインテグリンが集積したfocal adhesionが形成される。Rho-family低分子量G蛋白質の活性がこのような構造の形成や脱形成に深く関与していることは周知の通りである。また、最近、ARF-family低分子量G蛋白質がアクチン細胞骨格再構築に関与することも明らかにされている。ARF-family蛋白質は従来、主に細胞内での小胞輸送や膜輸送に関与することが示されていた分子である。
これまでに、インテグリン裏打ち蛋白質、パキシリンがゴルジ装置を含む核周辺部位にも局在していることを見い出していた。この局在がパキシリンのfilopodiaやlamellipodia構造への集積やインテグリン接着点形成過程に関連があると考え、生化学的/細胞生物学的解析を進めた。その結果、パキシリンが一連のARFGAP活性を持つ蛋白質と会合し、これらはパキシリンの核周辺局在とインテグリン接着点への集積、アクチン骨格構造制御、さらに、細胞運動性制御に関与することを明らかにした。
RacやCdc42の下流因子であるセリン/スレオニンリン酸化酵素PAK(p21-activated kinase)が、インテグリンシグナルや細胞骨格再構成に重要な役割を果たす事は良く知られている。調べた限りの幾つかの代表的な細胞接着斑蛋白質の中で、パキシリンのみが結合性を示した。PAKにはPIXやNck等、他に幾つかの結合蛋白質が知られているが、PAKとパキシリンとの結合はPAKとPIXやNckとの結合と競合的であった。さらに、パキシリンは不活性型及び、活性型PAKの両者に会合することができた。従って、パキシリンがPAKをfocal complexへアンカーする蛋白質であると考えられる。
日本学術振興会, 特定領域研究(C), (財)大阪バイオサイエンス研究所, 12218238 - インテグリン裏打ちタンパク質パキシリンの細胞内輸送過程
科学研究費助成事業
1999 - 2000
真崎 雄一
細胞の運動性や運動の方向性の制御は、細胞骨格構築の制御とインテグリン裏打ちタンパク質群の集積の制御との両者によって行われていると捉えることができる。本研究ではインテグリン裏打ちタンパク質群の一つであるパキシリンならびにその結合タンパク質に注目し、細胞の運動性及び細胞運動の方向性決定の基本機構を解明することを目指している。この目的を果たすため、本研究期間において、ARF1GAPの機能ドメインをもつパキシリン結合タンパク質(PAG1と命名)の局在部位の決定および機能解析を行った。
1.PAG1の局在部位の決定
申請者らは、PAG1がすでに蛍光抗体法を用いてゴルジ体にオーバーラップするように核周辺部に局在することを見出していたが、さらに電子顕微鏡を用いた抗体法においても同様の結果を得た。また、生化学的にも検討するために細胞を可溶化画分と膜画分に分けその局在を検討した結果、このタンパク質は、パキシリンとほぼ同様の割合(約20%)で膜画分に存在することがわかった。
2.PAG1の機能解析
PAG1の細胞内での役割を検討するために、繊維芽細胞にこのタンパク質あるいは、ARFに対してGAP活性を著しく低下させた変異体を強発現させ、細胞に与える影響を調べた。PAG1を強発現させた細胞では、バキシリンの細胞周縁部を除く細胞基質間接着点への集積の減少とアクチンストレスファイバーの減少がみられた。一方、GAP活性を著しく低下させた変異体では、そのような効果はみられなかった。さらに、この結果をテトラサイクリンによる発現調節系(Tet off system)を用いて検討した結果同様の結果が得られた。また、この系を用いて、細胞の接着性および運動性の変化を検討した結果、PAG1は、細胞の接着性にはほとんど影響を与えないものの、細胞の運動性に対して若干の促進活性を示した。
以上の結果をまとめ、Mol.Biol.Cell誌に発表した。
日本学術振興会, 奨励研究(A), (財)大阪バイオサイエンス研究所, 11780522 - 上皮細胞における細胞接着を介した生存性シグナルの解析
科学研究費助成事業
1999 - 1999
橋本 茂, 真崎 雄一, 佐邊 壽孝
上皮系細胞の生存性維持には増殖因子やサイトカインの刺激に加えて細胞接着シグナルが必要である。本研究では、インテグリン接着を介するシグナル伝達の機能的制御機構についてインテグリン裏打ちタンパク質パキシリンに着目した解析を進めている。今年度は、インテグリン接着点へのパキシリンの集積機構について解析を進めた。これまでに、パキシリンには核周辺領域に細胞質プールが存在し、運動中に細胞前方に能動的に集積させる機構が存在することを示唆させる結果を得ていた。この集積機構に関わる分子を解析するために、パキシリンに結合する分子群を同定し、想定される機能を持つ分子の検索を行った。その結果、ARF GAPモチーフを持つ一群のタンパク質を見い出した。その中の一つであるPAG3(Paxillin-associated ARF GAP protein 3)は、マクロファージ様に分化したU937細胞のcDNAからパキシリンをプローブとしたファーウエスタン法により見い出した。PAG3は、未分化の単球では細胞質に存在しているが、単球が接着性を獲得すると発現が亢進し、細胞辺縁部へ局在し、パキシリンと共局在することが観察された。また、PAG3のARF GAP活性がパキシリンの接着点への局在に重要な役割を果たしていること、分化した単球細胞の運動性の制御に関わっていることを示唆させる結果を得た。従って、パキシリンの細胞内局在は、単なる自由拡散ではなくARFの活性が関与する制御機構によって規定されていることが示された。また、ヒトの粥状動脈硬化病変においてマクロファージの特徴を示す泡沫化細胞にPAG3が強く発現していることを観察した。PAG3が病変の進展と関連した機能を有する可能性が示唆される。さらに、パキシリン及びその結合タンパク質がアクチン細胞骨格を制御する分子群と相互作用することを見い出した。細胞接着シグナルによる細胞骨格再構築の時間的/空間的制御機構に重点を置いた解析を開始している。
日本学術振興会, 特定領域研究(A), (財)大阪バイオサイエンス研究所, 11139271 - Role of paxillin isoforms and their tyrosine phosphorylation in cell migration.
Grants-in-Aid for Scientific Research
1998 - 1999
SABE Hisataka, MAZAKI Yuichi, YANO Hajime, HASHIMOTO Sigeru
Temporal and spatial regulation of actin-based cytoskeletal organization and focal adhesion formation play an essential role in cell migration. We found that tyrosine phosphorylation of paxillin and pi3OCas was a prominent event upon integrin activation during epithelial-mesenchymal transdifferentiation and cell migration. Tyrosine phosphorylation of p130^has been demonstrated to facilitate cell migration. We showed that tyrosine phosphorylation of paxillin cc acts to reduce haptotactic cell migration as well as transcellular invasive activities in several different experimental cell systems, whereas tyrosine phosphorylation of p130^ exerts opposing effects to those of paxillin
a. Each of the phosphorylation-null mutant acted as dominant-negatives for each phenotype. Moreover, we found that overexpression of paxillin a reduced the cell saturation density of NMuMG cells while overexpression of pi30^increased it. These effects also seemed to be dependent on the tyrosine phosphorylation events. Cell growth rates and morphologies at growing phases were not significantly altered, nor were cells transformed. Addition of epidermal growth factor increased saturation density of the paxillin α-overexpressing cells, while no further increment was observed in p130^ -overexpressing cells. We propose that tyrosine phosphorylation of paxillin a and p130^ exert opposing effects on several integrin-mediated cellular events, possibly through different signaling pathways. We also found that paxillin binds to several ARFGAPs. ARFGAPs are regulators of intracellular membrane trafficking. We currently analysing role of paxillin from aspects of its tyrosine phosphorylation and its interaction with ARFGAPs, in regulation of cell migratory activity.
Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (B)., OSAKA BIOSCIENCE INSTITUTE(OBI), 10480199 - 細胞接着制御因子パキシリンの細胞周期M期特異的変化の分子機構の解析
科学研究費助成事業
1998 - 1998
佐邊 壽孝, 真崎 雄一
インテグリンはその細胞質領域において多種類の蛋白質因子が集積し、この複合体を介してインテグリンのシグナル伝達は行われる。また、これらの複合体を介して細胞内の状況がインテグリンに伝わり、その結合性等が制御される。インテグリンと細胞外基質との接着は細胞の動的な運動過程において形成されるものであり、従って、このような動的過程においてどのようにしてインテグリンはそのシグナル伝達因子である集積蛋白質群と会合するのかは、細胞の運動性制御を明らかにする上で重要なポイントである。我々は、インテグリン裏打ち蛋白質パキシリンが細胞質においては、一部ゴルジ装置とオーバーラップする核周辺域の膜構造体に局在していることを明らかにした。パキシリンは可溶性蛋白質であることから、引き続き、パキシリンに結合する蛋白質の精製、単離同定を行ったところ、3種の新規蛋白質を見い出した。興味深いことにこれらは全てArfGAPに相当する配列を有しており、実際、in vitroではそのような活性を示すと考えられた。現在、細胞運動におけるパキシリンの細胞内輸送の分子機序を解明すべくその機能解析を進めている。
日本学術振興会, 萌芽的研究, (財)大阪バイオサイエンス研究所, 10878128
