Researcher Profile and Settings

Master

Affiliation (Master)

• Faculty of Medicine　Physiological Science　Pharmacology

Affiliation (Master)

• Faculty of Medicine　Physiological Science　Pharmacology

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Profile and Settings

Degree

• Ph.D.（The Graduate University for Advanced Studies）

Profile and Settings

• Name (Japanese)

  Mazaki

• Name (Kana)

  Yuichi

• Name

  200901032767342966

Achievement

Research Interests

• Neutrophil   Autoimmune disorders   infection   

Research Areas

• Life sciences / Pharmacology
• Life sciences / Allergies and connective tissue disease
• Life sciences / Infectious disease

Research Experience

• 2015 - Today 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

Published Papers

• 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 1347-8613 2023/12 [Refereed]
  
• 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 2023/10/30 [Refereed]
   

  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 2020/09 [Refereed][Not invited]
   

  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 2020/08 [Refereed][Not invited]
   

  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 Elsevier {BV} 513 (3) 708 - 713 2019/06/04 [Refereed][Not invited]
   

  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 2019/05 [Refereed][Not invited]
   

  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 0006-291X 2019/03/26 [Refereed][Not invited]
   

  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 0014-5793 2019/03 [Refereed][Not invited]
   

  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 0006-291X 2019/02/19 [Refereed][Not invited]
   

  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 2019 [Refereed][Not invited]
   

  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 Elsevier {BV} 126 (4) 527 - 532 1347-4421 2018/10 [Refereed][Not invited]
   

  Unsaturated carbonyl compounds, such as acrolein (ACR) and methyl vinyl ketone (MVK), are known as the environmental pollutants, and are contained in smoke, automobile exhaust, and heated oil. Although they can enter the circulation through the alveolar epithelium, the details of their effects on the vascular system remain to be clarified. We have recently reported that ACR and MVK induce protein kinase C (PKC) activation and cell damage mediated by intracellular Ca2+ in rat glioma cells (Higashi et al., J. Biosci. Bioeng., 124, 680-684, 2017). In this study, we have attempted to elucidate the effects of ACR and MVK on the vascular system, because blood vessels are easily exposed to these compounds. The rat aorta smooth muscle cells A7r5 were highly sensitive to ACR and MVK, whereas the human umbilical vein endothelial cells EA.hy926 were resistant to them. The ACR- and MVK-induced cell damage in A7r5 cells was PKC-dependent. In A7r5 cells, PKCα, PKCδ, PKCε, and PKCι were expressed. ACR and MVK induced PKCα and PKCδ translocation to the cell membrane. PKC activity was enhanced in A7r5 cells by ACR and MVK. These results indicate that the unsaturated carbonyl compounds might affect the vascular system by damaging smooth muscle cells via PKC activation.
• Molecular mechanism for ET-1-induced insulin resistance in skeletal muscle cells

  Takahiro Horinouchi, Yuichi Mazaki, Koji Terada, Soichi Miwa

  Folia Pharmacologica Japonica Japanese Pharmacological Society 151 (4) 140 - 147 0015-5691 2018 [Refereed][Invited]
   

  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 1389-1723 2017/12 [Refereed][Not invited]
   

  The unsaturated carbonyl compounds are known as the environmental pollutants. Acrolein (ACR) and methyl vinyl ketone (MVK) are representative unsaturated carbonyl compounds. ACR is contained in smoke, automobile exhaust, industrial waste, and several foods. MVK is widely used as the industrial chemical. Although ACR and MVK are highly toxic, the molecular mechanism for their cytotoxicity has been unclear. We have previously reported that ACR and MVK are major cytotoxic compounds in the gas phase of cigarette smoke, and protein kinase C (PKC) inhibitor and NADPH oxidases inhibitor partially rescued cells from ACR- or MVK-induced cell death (Noya et al., Toxicology, 314, 1-10, 2013). PKC translocation, which is hallmark for PKC activation, and cell damage were induced by treatment of cultured cells with ACR or MVK. Intracellular Ca2+ chelator completely suppressed ACR- or MVK-induced PKC translocation to the cell membrane and cell damage, while extracellular Ca2+ chelator had no effects on ACR- and MVK-induced cytotoxicity. These results suggest that intracellular Ca2+ is an essential factor for cell damage caused by both PKC-dependent and PKC-independent pathways, and mobilization of Ca2+ from intracellular Ca2+ stores is induced by ACR or MVK.
• 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 1478-811X 2017/10/02 [Refereed][Not invited]
   

  BACKGROUND: The small GTPase ARF1 mediates membrane trafficking mostly from the Golgi, and is essential for the G protein-coupled receptor (GPCR)-mediated chemotaxis of neutrophils. In this process, ARF1 is activated by the guanine nucleotide exchanger GBF1, and is inactivated by the GTPase-activating protein GIT2. Neutrophils generate the Gβγ-PAK1-αPIX-GIT2 linear complex during GPCR-induced chemotaxis, in which αPIX activates RAC1/CDC42, which then employs PAK1. However, it has remained unclear as to why GIT2 is included in this complex. RESULTS: We investigated the association between ARF1 and RAC1/CDC42 during the fMLP-stimulated chemotaxis of HL60 cells. We found that the silencing of GBF1 significantly impaired the recruitment of RAC1 to the leading edges, but not PAK1, αPIX, RAC2, or CDC42. A significant population of RAC1 colocalized with ARF1 at the leading edges in stimulated cells, whereas fMLP activated both ARF1 and ARF5. Consistently, the silencing of ARF1, but not ARF5, impaired the recruitment of RAC1, whereas the silencing of RAC1 did not affect the recruitment of ARF1 to the leading edges. CONCLUSIONS: Our results indicated that the activation of ARF1 triggers the plasma membrane recruitment of RAC1 in GPCR-mediated chemotaxis, which is essential for cortical actin remodeling. Thus, membrane remodeling at the leading edges appears to precede actin remodeling in chemotaxis. Together with the fact that GIT2, which inactivates ARF1, is an integral component of the machinery activating RAC1, we proposed a model in which the ARF1-RAC1 linkage enables the regulation of ARF1 by repetitive on/off cycles during GPCR-mediated neutrophil chemotaxis.
• 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 0007-1188 2016/03 [Refereed][Not invited]
   

  BACKGROUND AND PURPOSE: Endothelin-1 (ET-1) reduces insulin-stimulated glucose uptake in skeletal muscle, inducing insulin resistance. Here, we have determined the molecular mechanisms underlying negative regulation by ET-1 of insulin signalling. EXPERIMENTAL APPROACH: We used the rat L6 skeletal muscle cells fully differentiated into myotubes. Changes in the phosphorylation of Akt was assessed by Western blotting. Effects of ET-1 on insulin-stimulated glucose uptake was assessed with [(3) H]-2-deoxy-d-glucose ([(3) H]2-DG). The C-terminus region of GPCR kinase 2 (GRK2-ct), a dominant negative GRK2, was overexpressed in L6 cells using adenovirus-mediated gene transfer. GRK2 expression was suppressed by transfection of the corresponding short-interfering RNA (siRNA). KEY RESULTS: In L6 myotubes, insulin elicited sustained Akt phosphorylation at Thr(308) and Ser(473) , which was suppressed by ET-1. The inhibitory effects of ET-1 were prevented by treatment with a selective ETA receptor antagonist and a Gq protein inhibitor, overexpression of GRK2-ct and knockdown of GRK2. Insulin increased [(3) H]2-DG uptake rate in a concentration-dependent manner. ET-1 noncompetitively antagonized insulin-stimulated [(3) H]2-DG uptake. Blockade of ETA receptors, overexpression of GRK2-ct and knockdown of GRK2 prevented the ET-1-induced suppression of insulin-stimulated [(3) H]2-DG uptake. In L6 myotubes overexpressing FLAG-tagged GRK2, ET-1 facilitated the interaction of endogenous Akt with FLAG-GRK2. CONCLUSIONS AND IMPLICATIONS: Activation of ETA receptors with ET-1 suppressed insulin-induced Akt phosphorylation at Thr(308) and Ser(473) and [(3) H]2-DG uptake in a GRK2-dependent manner in skeletal muscle cells. These findings suggest that ETA receptors and GRK2 are potential targets for overcoming insulin resistance.
• 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 0918-6158 2016 [Refereed][Not invited]
   

  Cigarette mainstream smoke is composed of gas and tar phases and contains >4000 chemical constituents, including nicotine and tar. The substances in the gas phase but not in the tar phase can pass through the airway epithelial barrier, enter the systemic circulation via the pulmonary circulation, and increase systemic oxidative damage, leading to the development of cigarette smoking-related diseases such as atherosclerosis. Recently, we identified some stable carbonyl compounds, including acrolein (ACR) and methyl vinyl ketone (MVK), as major cytotoxic factors in nicotine- and tar-free cigarette smoke extract (CSE) of the gas phase. CSE, ACR, and MVK induce protein kinase C (PKC)-dependent activation of reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) and subsequent generation of reactive oxygen species (ROS) via NOX, causing plasma membrane damage and cell apoptosis. CSE, ACR, and MVK also trigger carbonylation of PKC, which is an irreversible oxidative modification. Cell damage and PKC carbonylation in response to treatment with CSE, ACR, or MVK are abolished by thiol-containing antioxidants such as N-acetyl-L-cysteine and reduced glutathione. Thus pharmacological modulation of PKC and NOX activities and the trapping of ROS are potential strategies for the prevention of diseases related to cigarette smoking.
• 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 0918-6158 2016 [Refereed][Not invited]
   

  The gas phase of cigarette smoke is important from the viewpoint of human health, because it can pass through alveolar epithelium and enter the circulation. There is no standard method for the preparation of a gas phase extract of cigarette smoke (CSE), although CSE is widely used for research instead of whole cigarette smoke. We have established a standard method for the preparation of CSE. One cigarette per trial is continuously combusted under a reduced pressure generated by an aspiration pump with a velocity of 1.050 L/min: the main stream of the smoke is passed through a Cambridge filter to remove tar, and subsequently, bubbled through a glass ball filter (pore size, 20-30 µm) into 15 mL of phosphate-buffered saline (PBS). To express the concentration of CSE, a virtual tar concentration is introduced, which is calculated assuming that tar trapped on the Cambridge filter is dissolved in the PBS. CSEs prepared from smaller numbers of cigarettes (original virtual tar concentration≤15 mg/mL) show similar concentration-response curves for cytotoxicity versus virtual tar concentrations. CSEs prepared from various brands of cigarettes and by different smoking regimes (continuous and puff smoking) show similar cytotoxic potency if the virtual tar concentrations are the same. In conclusion, using the standardized method for CSE preparation in combination with the virtual tar concentration, it becomes possible to simply and rapidly prepare standard CSEs with defined concentrations from any brand of cigarettes, which are toxicologically equivalent to CSE prepared by puff smoking.
• 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  1932-6203 2016 [Refereed][Not invited]
   

  BACKGROUND: Accumulating evidence has shown that methionine- and choline-deficient high fat (MCD+HF) diet induces the development of nonalcoholic fatty liver disease (NAFLD), in which elevated reactive oxygen species play a crucial role. We have reported that peroxiredoxin 4 (PRDX4), a unique secretory member of the PRDX antioxidant family, protects against NAFLD progression. However, the detailed mechanism and potential effects on the intestinal function still remain unclear. METHODS & RESULTS: Two weeks after feeding mice a MCD+HF diet, the livers of human PRDX4 transgenic (Tg) mice exhibited significant suppression in the development of NAFLD compared with wild-type (WT) mice. The serum thiobarbituric acid reactive substances levels were significantly lower in Tg mice. In contrast, the Tg small intestine with PRDX4 overexpression showed more suppressed shortening of total length and villi height, and more accumulation of lipid in the jejunum, along with lower levels of dihydroethidium binding. The enterocytes exhibited fewer apoptotic but more proliferating cells, and inflammation was reduced in the mucosa. Furthermore, the small intestine of Tg mice had significantly higher expression of cholesterol absorption-regulatory factors, including liver X receptor-α, but lower expression of microsomal triglyceride-transfer protein. CONCLUSION: Our present data provide the first evidence of the beneficial effects of PRDX4 on intestinal function in the reduction of the severity of NAFLD, by ameliorating oxidative stress-induced local and systemic injury. We can suggest that both liver and intestine are spared, to some degree, by the antioxidant properties of PRDX4.
• 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 2193-1801 2016 [Refereed][Not invited]
   

  BACKGROUND: Multiple sclerosis (MS) is an autoimmune disease characterized by inflammatory demyelination, gliosis and axonal loss in the Central Nervous System. Although the etiology of the disease has remained enigmatic, recent studies have suggested a role of the innate-like T cells, called Mucosal Associated Invariant T cells (MAITs) in the pathophysiology. In the present study, we have analyzed the relative frequency of MAITs and the expression of the cell surface antigens in MAITs to seek a possible link to the disease. RESULTS: There was little difference in the frequency of total MAITs between healthy donors (HDs) and untreated MS patients, whereas the latter harbored more CD8(lo/neg) (DN) MAITs concomitant with a decrease in CD8(high) MAITs and in CD4 MAITs compared with those in HDs. While the expression of CCR5, CCR6, CD95, CD127, and CD150 has increased in untreated subjects compared with that in HDs, CD45RO has declined in untreated subjects in both DN MAITs and CD8(hi) MAITs. FTY720 therapy has increased the relative frequency of total MAITs in a time-dependent fashion up to 2 years. Intriguingly, FTY720 therapy for 3 years reversed the above phenotype, engendering more CD8(high) MAITs accompanied with decreased DN MAITs. FTY720 therapy affected the cytokine production from CD4 T cells and also enhanced the relative frequency of cells producing both TNF-α and IFN-γ from MAITs, CD8 T cells, and CD4 T cells compared with that in untreated subjects. CONCLUSIONS: FTY 720 therapy enhanced the relative frequency of MAITs in MS patients in a time-dependent manner. Although the expression of CD8 in MAITs has been affected early by FTY720, longer treatment has reversed the phenotypic change. These data demonstrated that FTY720 induced dynamic change in the relative frequency and in the phenotype of MAITs in MS.
• 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 1059-1524 2012/07 [Refereed][Not invited]
   

  Most chemoattractants for neutrophils bind to the Gα(i) family of heterotrimeric G protein-coupled receptors (GPCRs) and release Gβγ subunits to activate chemotaxis and superoxide production. GIT2, a GTPase-activating protein for Arf1, forms a complex with Gβγ and is integral for directional sensing and suppression of superoxide production. Here we show that GBF1, a guanine nucleotide exchanging factor for Arf-GTPases, is primarily responsible for Arf1 activation upon GPCR stimulation and is important for neutrophil chemotaxis and superoxide production. We find that GBF1 bears a novel module, namely binding to products of phosphatidyl inositol 3-kinase (PI3K), which binds to products of PI3Kγ. Through this binding, GBF1 is translocated from the Golgi to the leading edge upon GPCR stimulation to activate Arf1 and recruit p22phox and GIT2 to the leading edge. Moreover, GBF1-mediated Arf1 activation is necessary to unify cell polarity during chemotaxis. Our results identify a novel mechanism that links PI3Kγ activity with chemotaxis and superoxide production in GPCR signaling.
• 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 1059-1524 2008/03 [Refereed][Not invited]
   

  The small GTP-binding protein Arf6 regulates membrane remodeling at cell peripheries and plays crucial roles in higher orders of cellular functions including tumor invasion. Here we show that Fbx8, an F-box protein bearing the Sec7 domain, mediates ubiquitination of Arf6. This ubiquitination did not appear to be linked to immediate proteasomal degradation of Arf6, whereas Fbx8 knockdown caused hyperactivation of Arf6. Expression of Fbx8 protein was substantially lost in several breast tumor cell lines, in which Arf6 activity is pivotal for their invasion. Forced expression of Fbx8 in these cells suppressed their Arf6 activities and invasive activities, in which the F-box and Sec7 domains of Fbx8 are required. Together with the possible mechanism as to how Fbx8-mediated ubiquitination interferes with the functions of Arf6, we propose that Fbx8 provides a novel suppressive control of Arf6 activity through noncanonical ubiquitination. Our results indicate that dysfunction of Fbx8 expression may contribute to the invasiveness of some breast cancer cells.
• 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 1465-7392 2008/01 [Refereed][Not invited]
   

  Epidermal growth factor (EGF) receptor (EGFR) signalling is implicated in tumour invasion and metastasis. However, whether there are EGFR signalling pathways specifically used for tumour invasion still remains elusive. Overexpression of Arf6 and its effector, AMAP1, correlates with and is crucial for the invasive phenotypes of different breast cancer cells. Here we identify the mechanism by which Arf6 is activated to induce tumour invasion. We found that GEP100/BRAG2, a guanine nucleotide exchanging factor (GEF) for Arf6, is responsible for the invasive activity of MDA-MB-231 breast cancer cells, whereas the other ArfGEFs are not. GEP100, through its pleckstrin homology domain, bound directly to Tyr1068/1086-phosphorylated EGFR to activate Arf6. Overexpression of GEP100, together with Arf6, caused non-invasive MCF7 cells to become invasive, which was dependent on EGF stimulation. Moreover, GEP100 knockdown blocked tumour metastasis. GEP100 was expressed in 70% of primary breast ductal carcinomas, and was preferentially co-expressed with EGFR in the malignant cases. Our results indicate that GEP100 links EGFR signalling to Arf6 activation to induce invasive activities of some breast cancer cells, and hence may contribute to their metastasis and malignancy.
• 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 0261-4189 2007/02/07 [Refereed][Not invited]
   

  Expression of AMAP1 correlates well with the invasive phenotypes and malignancy of human primary breast carcinomas. AMAP1 recruits its binding proteins, such as cortactin and paxillin, to sites of Arf6 activation to form invadopodia. A mouse ortholog of AMAP1, ASAP1, is known to bind to CIN85, a binding partner of an E3 ligase, Cbl. Here, we found that CIN85 colocalizes with AMAP1 at invadopodia, and binding of AMAP1 with CIN85 is important for the invasive activities of breast cancer cells, including MDA-MB-231. siRNA-mediated silencing of CIN85, as well as Cbl, also inhibited the invasion. We moreover found that AMAP1 is monoubiquitinated, rather than polyubiquitinated, by virtue of Cbl and provide evidence that the ability of AMAP1 to be monoubiquitinated is important for its involvement in invasion. Our results indicate that CIN85, as well as Cbl, which is a well-known suppressor of growth factor receptor signaling, can be positively involved in tumor invasion, and suggest that a complex epigenetic process is involved in AMAP1 function in breast cancer cell invasion.
• 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 0955-0674 2006/10 [Refereed][Not invited]
   

  The identification of several ArfGAP proteins as binding partners of paxillin, an integrin signaling and scaffolding protein, has suggested the existence of molecular links between integrin functions and intracellular traffic, as proposed by MS Bretscher long ago. Among the paxillin-binding ArfGAPs, AMAP1 has recently been strongly implicated in tumor invasion as well as malignancy, owing to its highly augmented expression in tumors and its direct involvement in invasive activities. Another ArfGAP, Git2, was found to be a component of the Gbetagamma-mediated directional sensing machinery, while simultaneously playing an essential role in the suppressive control of superoxide production, which is mediated by vesicle transport in GPCR-stimulated neutrophils. These emerging molecular mechanisms may further delineate key processes regulating intracellular traffic as principal controls of cell motility and invasive activities.
• 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 1529-2908 2006/07 [Refereed][Not invited]
   

  In neutrophils, superoxide anion production generally accompanies chemotaxis and functions in killing invading pathogens. The GIT2 GTPase-activating protein binds to the guanine nucleotide-exchange factor alphaPIX. Here we show that GIT2 was necessary for directional chemotaxis and for the suppression of superoxide production in G protein-coupled receptor-stimulated neutrophils. GIT2 was also necessary for the orientation of superoxide production toward chemoattractant sources. GIT2 suppressed the activity of ADP ribosylation factor 1 and was a component of the Gbetagamma subunit-mediated direction-sensing machinery 'downstream' of G protein-coupled receptor signaling. This study establishes a function for GIT2 in linking chemotaxis and superoxide production in neutrophils and shows that loss of GIT2 in vivo leads to an immunodeficient state.
• 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 0261-4189 2005/03/09 [Refereed][Not invited]
   

  Identification of the molecular machinery employed in cancer invasion, but not in normal adult cells, will greatly contribute to cancer therapeutics. Here we found that an ArfGAP, AMAP1/PAG2, is expressed at high levels in highly invasive breast cancer cells, but at very low levels in noninvasive breast cancer cells and normal mammary epithelial cells. siRNA-mediated silencing of AMAP1 effectively blocked the invasive activities. AMAP1 expression in human breast primary tumors also indicated its potential correlation with malignancy. Paxillin and cortactin have been shown to colocalize at invadopodia and play a pivotal role in breast cancer invasion. We found that AMAP1 is also localized at invadopodia, and acts to bridge paxillin and cortactin. This AMAP1-mediated trimeric protein complex was detected only in invasive cancer cells, and blocking this complex formation effectively inhibited their invasive activities in vitro and metastasis in mice. Our results indicate that AMAP1 is a component involved in invasive activities of different breast cancers, and provide new information regarding the possible therapeutic targets for prevention of breast cancer invasion and metastasis.
• 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 Rockefeller University Press 166 (2) 283 - 95 0021-9525 2004/07/19 [Refereed][Not invited]
   

  Integrins can intercommunicate with cadherins. Here, we examined their possible relationship by use of small interfering RNA-mediated protein knockdown in HeLa cells. We found that a subset of integrin signaling molecules, namely Fak and paxillin, but not p130 Crk-associated substrate or proline-rich tyrosine kinase 2, participate in processes regulating N-cadherin-based cell-cell adhesion. Paxillin was found to be required primarily for the recruitment of Fak to robust focal adhesions. Our results suggest that at least some signals involving Fak are linked to a mechanism down-regulating Rac1 activity at the cell periphery, which appears to be important for the formation of N-cadherin-based adhesions in motile cells. Our analyses simultaneously exemplified the essential role of Fak in the maintenance of cell-cell adhesions in collective cell migration, a type of migration occurring in embryonic development and carcinoma invasion.
• 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 0021-9525 2002/11/25 [Refereed][Not invited]
   

  RhoA activity is transiently inhibited at the initial phase of integrin engagement, when Cdc42- and/or Rac1-mediated membrane spreading and ruffling predominantly occur. Paxillin, an integrin-assembly protein, has four major tyrosine phosphorylation sites, and the phosphorylation of Tyr31 and Tyr118 correlates with cell adhesion and migration. We found that mutation of Tyr31/118 caused enhanced activation of RhoA and premature formation of stress fibers with substantial loss of efficient membrane spreading and ruffling in adhesion and migration of NMuMG cells. These phenotypes were similar to those induced by RhoA(G14V) in parental cells, and could be abolished by expression of RhoA(T19N), Rac1(G12V), or p190RhoGAP in the mutant-expressing cells. Phosphorylated Tyr31/118 was found to bind to two src homology (SH)2 domains of p120RasGAP, with coprecipitation of endogenous paxillin with p120RasGAP. p190RhoGAP is known to be a major intracellular binding partner for the p120RasGAP SH2 domains. We found that Tyr31/118-phosphorylated paxillin competes with p190RhoGAP for binding to p120RasGAP, and provides evidence that p190RhoGAP freed from p120RasGAP efficiently suppresses RhoA activity during cell adhesion. We conclude that Tyr31/118-phosphorylated paxillin serves as a template for the localized suppression of RhoA activity and is necessary for efficient membrane spreading and ruffling in adhesion and migration of NMuMG cells.
• 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 0021-9258 2002/02/22 [Refereed][Not invited]
   

  Using mass spectrometry we have identified proteins which co-immunoprecipitate with paxillin, an adaptor protein implicated in the integrin-mediated signaling pathways of cell motility. A major component of paxillin immunoprecipitates was poly(A)-binding protein 1, a 70-kDa mRNA-binding protein. Poly(A)-binding protein 1 associated with both the alpha and beta isoforms of paxillin, and this was unaffected by RNase treatment consistent with a protein-protein interaction. The NH(2)-terminal region of paxillin (residues 54-313) associated directly with poly(A)-binding protein 1 in cell lysates, and with His-poly(A)-binding protein 1 immobilized in microtiter wells. Binding was specific, saturable and of high affinity (K(d) of approximately 10 nm). Cell fractionation studies showed that at steady state, the bulk of paxillin and poly(A)-binding protein 1 was present in the "dense" polyribosome-associated endoplasmic reticulum. However, inhibition of nuclear export with leptomycin B caused paxillin and poly(A)-binding protein 1 to accumulate in the nucleus, indicating that they shuttle between the nuclear and cytoplasmic compartments. When cells migrate, poly(A)-binding protein 1 colocalized with paxillin-beta at the tips of lamellipodia. Our results suggest a new mechanism whereby a paxillin x poly(A)-binding protein 1 complex facilitates transport of mRNA from the nucleus to sites of protein synthesis at the endoplasmic reticulum and the leading lamella during cell migration.
• 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 Rockefeller University Press 193 (8) 955 - 966 0022-1007 2001/04 [Refereed][Not invited]
   

  The Fc gamma receptor (Fc gammaR)-mediated phagocytosis of macrophages is a complex process where remodeling of both the actin-based cytoskeleton and plasma membrane occur coordinately. Several different families of small GTPases are involved. We have isolated a GTPase-activating protein (GAP) for ADP-ribosylation factor (ARF), paxillin-associated protein with ARFGAP activity (PAG)3/Pap alpha /KIAA0400, from mature monocytes and macrophage-like cells. Mam malian ARFs fall into three classes, and the class III isoform (ARF6) has been shown to be involved in Fc gammaR-mediated phagocytosis. Here we report that PAG3 is enriched together with ARF6 and F-actin at phagocytic cups formed beneath immunoglobulin G-opsonized beads in P388D1 macrophages, in which overexpression of ARF6, but not ARF1 (class I) or ARF5 (class II), inhibits the phagocytosis. Overexpression of PAG3, but riot its GAP-inactive mutant, attenuated the focal accumulation of F-actin and blocked phagocytosis, although surface levels of the Fc gamma Rs were not affected. Other ubiquitously expressed ARFGAPs, G protein-coupled receptor kinase interactors GIT2 and GIT2-short/KIAA0148, which we have shown to exhibit GAP activity for ARF1 in COS-7 cells, did not accumulate at the phagocytic cups or inhibit phagocytosis. Moreover, cooverexpression of ARF6, but not ARF1 or ARF5, restored the phagocytic activity of PAG3-overexpressing cells. We propose that PAG3 acts as a GAP for ARF6 and is hence involved in Fc gammaR-mediated phagocytosis in mouse macrophages.
• 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 1059-1524 2001/03 [Refereed][Not invited]
   

  Paxillin acts as an adaptor protein in integrin signaling. We have shown that paxillin exists in a relatively large cytoplasmic pool, including perinuclear areas, in addition to focal complexes formed at the cell periphery and focal adhesions formed underneath the cell. Several ADP-ribosylation factor (ARF) GTPase-activating proteins (GAPs; ARFGAPs) have been shown to associate with paxillin. We report here that Git2-short/KIAA0148 exhibits properties of a paxillin-associated ARFGAP and appears to be colocalized with paxillin, primarily at perinuclear areas. A fraction of Git2-short was also localized to actin-rich structures at the cell periphery. Unlike paxillin, however, Git2-short did not accumulate at focal adhesions underneath the cell. Git2-short is a short isoform of Git2, which is highly homologous to p95PKL, another paxillin-binding protein, and showed a weaker binding affinity toward paxillin than that of Git2. The ARFGAP activities of Git2 and Git2-short have been previously demonstrated in vitro, and we provided evidence that at least one ARF isoform, ARF1, is an intracellular substrate for the GAP? activity of Git2-short. We also showed that Git2-short could antagonize several known ARF1-mediated phenotypes: overexpression of Git2-short, but not its GAF-inactive mutant, caused the redistribution of Golgi protein beta -COP and reduced the amounts of paxillin-containing focal adhesions and actin stress fibers. Perinuclear localization of paxillin, which was sensitive to ARF inactivation, was also affected by Git2-short overexpression. On the other hand, paxillin localization to focal complexes at the cell periphery was unaffected or even augmented by Git2-short overexpression. Therefore, an ARFGAP protein weakly interacting with paxillin, Git2-short, exhibits pleiotropic functions involving the regulation of Golgi organization, actin cytoskeletal organization, and subcellular localization of paxillin, all of which need to be coordinately regulated during integrin-mediated cell adhesion and intracellular signaling.
• 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 0021-9258 2001/02/23 [Refereed][Not invited]
   

  p21-activated kinases (PAKs) are implicated in integrin signalings, and have been proposed to associate with paxillin indirectly. We show here that paxillin can bind directly to PAK3. We examined several representative focal adhesion proteins, and found that paxillin is the sole protein that associates with PAK3. PAK3 associated with the α and β isoforms of paxillin, but not with γ. We also show that paxillin α associated with both the kinase-inactive and the Cdc42-activated forms of PAK3 in vivo, without affecting the activation states of the kinase. A number of different functions have been ascribed to PAKs and PAKs can bind directly to growth factor signaling-adaptor molecule, Nck, anda and a guanine nucleotide exchanger, βPIX. Our results revealed that paxillin α can compete with Nck and βPIX in the binding of PAK3. Moreover, paxillin α can be phosphorylated by PAK3 at serine. Therefore, paxillin α but not γ, appears to be capable of linking both the kinase-inactive and activated forms of PAK3 to integrins independent of Nck and βPIX, as Nck links PAK1 to growth factor receptors. Our results also revealed that paxillin is involved in highly complexed protein-protein interactions in integrin signaling.
• 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 American Society for Cell Biology ({ASCB}) 11 (4) 1315 - 1327 1059-1524 2000/04 [Refereed][Not invited]
   

  Paxillin acts as an adaptor molecule in integrin signaling. Paxillin is localized to focal contacts but seems to also exist in a relatively large cytoplasmic pool. Here, we report the identification of a new paxillin-binding protein, FAGS (paxillin-associated protein with ADP-ribosylation factor [ARF] GTPase-activating protein [GAP] activity, number 3), which is involved in regulation of the subcellular localization of paxillin. FAGS bound to all paxillin isoforms and was induced during monocyte maturation, at which time paxillin expression is also increased and integrins are activated. PAG3 was diffusely distributed in the cytoplasm in premature monocytes but became localized at cell periphery in mature monocytes, a fraction of which then colocalized with paxillin. PAG3, on the other hand, did not accumulate at focal adhesion plaques, suggesting that PAG3 is not an integrin assembly protein. PAG3 was identical to KIAA0400/Pap alpha, which was previously identified as a Pyk2-binding protein bearing a GAP activity toward several ARFs in vitro. Mammalian ARFs fall into three classes, and we showed that all classes could affect subcellular localization of paxillin. We also examined possible interaction of FAGS with ARFs and showed evidence that at least one of them, ARF6, seems to be an intracellular substrate for GAP activity of FAGS. Moreover, overexpression of PAG3, but not its GAP-inactive mutant, inhibited paxillin recruitment to focal contacts and hampered cell migratory activities, whereas cell adhesion activities were almost unaffected. Therefore, our results demonstrate that paxillin recruitment to focal adhesions is not mediated by simple cytoplasmic diffusion; rather, PAG3 appears to be involved in this process, possibly through its GAP activity toward ARF proteins. Our result thus delineates a new aspect of regulation of cell migratory activities.
• 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 0261-4189 2000/02 [Refereed][Not invited]
   

  Both F10 and BL6 sublines of B16 mouse melanoma cells are metastatic after intravenous injection, but only BL6 cells are metastatic after subcutaneous injection, Retrotransposon insertion was found to produce an N-terminally truncated form (Delta gamma 1) of the B56 gamma 1 regulatory subunit isoform of protein phosphatase (PP) 2A in BL6 cells, but not in F10 cells, We found an interaction of paxillin with PP2A C and B56 gamma subunits by co-immunoprecipitation. B56 gamma 1 co-localized with paxillin at focal adhesions, suggesting a role for this isoform in targeting PP2A to paxillin, In this regard, Delta gamma 1 behaved similarly to B56 gamma 1. However, the Delta gamma 1-containing PP2A heterotrimer was insufficient for the dephosphorylation of paxillin. Transfection with Delta gamma 1 enhanced paxillin phosphorylation on serine residues and recruitment into focal adhesions, and cell spreading with an actin network. In addition, Delta gamma 1 rendered F10 cells as highly metastatic as BL6 cells. These results suggest that mutations in PP2A regulatory subunits may cause malignant progression.
• 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 0021-9258 1998/08 [Refereed][Not invited]
   

  Paxillin, a focal adhesion protein, exists as multiple isoforms in humans (alpha, beta, and gamma), To understand more about the physiological role of each isoform, we have employed the mouse system. We found that although the alpha and beta isoforms are present in the mouse, the gamma isoform is not. The alpha isoform protein was detected clearly in most adult tissues, whereas the beta isoform protein was almost undetectable except in spleen, testis, thymus, and lung. On the other hand, mRNAs of both isoforms were detectable in all tissues we examined. High levels of the beta isoform protein was detected in peritoneal exudate macrophage cells in adult mouse as well as in cultured fibroblasts, together with the alpha isoform, The alpha isoform was expressed at a constant level throughout the embryonic stages we examined, whereas the beta isoform protein was detected at the mid-stages of development and increased to levels almost equal to those of the a isoform during the late stages of embryogenesis, Therefore, unlike the alpha isoform, expression of the beta isoform protein is restricted in adult tissues. Moreover, we showed that alpha and beta isoforms were colocalized within the same focal adhesion plaques, and cytoplasmic pools of both isoforms exist in the perinuclear area, colocalized with the Gels apparatus.
• 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 0012-1606 1998/01 [Refereed][Not invited]
   

  Mitf encodes a basic helix-loop-helix-leucine-zipper (bHLHzip) protein that is known to function in the development of melanocytes, pigmented epithelial cells (PECs), osteoclasts, and mast cells. In this paper, we report on the isolation, expression, and overexpression of the chicken Mitf and discuss the role of its protein product in the differentiation and transdifferentiation of PECs. Northern blotting showed that chicken Miff is predominantly expressed in embryonic retinal pigmented epithelium (PE), but is expressed at low levels in other tissues. A 5' RACE analysis revealed differences in the 5' region of Mitf mRNA in PE and other tissues. Immunological analysis revealed that Mitf, the protein encoded by Miff, is first detected in the nuclei of the optic vesicle cells at embryonic stage 13 in a restricted region covered with mesenchymal cells. From stage 14 to 24, the specific staining is observable in the PE and precursor of the PE, the outer layer of the optic cup. In embryos at stages later than stage 25, the signals for Mitf in the future iris, ciliary body, and posterior retinal regions become faint. These results show that expression of Mitf starts at the optic vesicle stage at which no other marker genes for PECs such as mmp115 and tyrosinase are expressed. Dedifferentiation of cultured retinal PECs (rPECs) was induced by phenylthiourea and testicular hyaluronidase, bFGF, or TGF-beta. Miff expression was inhibited by these factors and reactivated during redifferentiation of the dedifferentiated cells into rPECs, showing the correlation between Miff expression and rPEC differentiation. Retrovirus-mediated overexpression of Miff inhibited bFGF-induced dedifferentiation and transdifferentiation of rPECs to both lens and neural cells. These findings showed that downregulation of Miff expression is essential for the transdifferentiation of rPEC. Miff overexpression caused hyperpigmentation in cultured rPECs and suppressed the changes in gene expression induced by bFGF. Miff overexpression promoted expression of mmp115 and tyrosinase in bFGF-treated rPECs suggesting a critical role for Mitf in rPEC differentiation. Miff overexpression, however, did not promote expression of another rPEC-specific gene, pP344, in bFGF-treated rPECs. This result suggests the presence of other regulatory genes promoting rPEC differentiation. The expression patterns of pax6 and Mitf are complementary both in vivo and in vitro. Overexpression of Miff inhibited expression of pax6, in cultured rPECs. These observations suggest that Mitf regulates pax6 expression negatively. (C) 1998 Academic Press.
• 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 0950-9232 1997/10 [Refereed][Not invited]
   

  Mitotic cells typically lack well-formed focal adhesions, As an approach to explore the dynamic process regulating the focal adhesion assembly, we examined states of focal adhesion proteins during mitosis of the cell cycle. We found that the amount of paxillin was significantly reduced during mitosis of the cell cycle, whereas other focal adhesion proteins including talin, vinculin and Focal Adhesion Kinase did not. Proteolytic degradation appeared to be involved in the mitotic reduction, but transcriptional and/or translational controls of the mRNA were not essential for this downregulation. Moreover, concurrent with the decreased protein level, phosphorylation status of paxillin altered during mitosis; mitotic paxillin was phosphorylated primarily on serine and dephosphorylated on tyrosine while interphase one was phosphorylated both on serine and tyrosine. We found that mitotic phosphorylation created an electrophoretically slow-migrating population of paxillin which was barely detected in interphase cells. This mitotic specific modification occurred with both alpha and beta isoforms of paxillin. We also examined the fate of paxillin protein by changing its protein amount. We found that majority of paxillin overexpressed was subjected to the specific modification but not to the downregulation in the mitotic arrested cells. On the other hand, paxillin exogenously expressed at a moderate level was subjected to both the mitotic modification and downregulation. Collectively, we concluded that paxillin's specific serine phosphorylation together with the proteolytic downregulation of a limited fraction of paxillin is taken place during the mitosis of the cell cycle.
• 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 0021-9258 1997/03 [Refereed][Not invited]
   

  The versatility of integrin functions is mediated by engagement of a number of proteins that assemble with integrins. Among them, paxillin is one of the important molecules interacting with a variety of signaling molecules and cytoskeletal building blocks. We report here that paxillin is not a single molecule with a unique physiological property. We identified two human paxillin isoforms, beta and gamma. These isoforms have distinct amino acid insertions; each consists of a distinct exon, at the same site of previously reported paxillin (paxillin alpha). Several proteins were co-precipitated with paxillin, and we found that beta bound to focal adhesion kinase but weakly to vinculin, and gamma bound to vinculin but only weakly to focal adhesion kinase, although both bound equally to talin. No additional proteins were found to bind to beta and gamma over those binding to alpha. Unlike the alpha isoform, beta and gamma mRNAs were not detected in normal tissues, but several cancer cells expressed both alpha and beta proteins simultaneously. All three isoform proteins were expressed in promonocytic cells with ratios comparable with each other, and the expression patterns were altered during differentiation of floating promonocytic cells into adherent macrophage-like cells. Therefore, each isoform of paxillin exhibits distinct expression and different biochemical as well as physiological properties and thereby appears to act as a distinct module involved in different functions of integrins.
• 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 0012-1592 1996/08 [Refereed][Not invited]
   

  When retinal pigmented epithelial cells (PEG) of chick embryos are cultured under appropriate conditions, the phenotype changes to that of lens cells through a process known as transdiiferentiation. The first half of the process, characterized by dedifferentiation of PEG, is accompanied by a marked decrease in adhesiveness of PEC to collagen type I- or type IV-coated dishes. To understand the underlying mechanisms of this change, we analyzed the expression of integrins, which are major receptors for extracellular matrix components. Northern blot analysis with cDNA probes for chicken alpha 3, alpha 6, alpha 8, alpha v, beta 1 and beta 5 integrin mRNA showed that the genes for all these integrins are transcribed at similar levels in PEC and dedifferentiated PEC (dePEC). Further analysis of beta 1 integrin, which is a major component of integrin heterodimers, showed that although the protein amount of beta 1 integrin was not changed, its localization at focal contacts seen in PEC was lost in dePEC. When anti-beta 1 integrin antibody was added to the PEC culture medium, a decrease of cell-substrate adhesiveness occurred, followed by a gradual change in both morphology and gene expression patterns to ones similar to those of dePEC. These findings suggest that an appropriate distribution of beta 1 integrin plays an essential role in maintaining the differentiated state of PEC through cell-substrate adhesion.

MISC

• 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.
• 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.
• 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.
• 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.
• 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  [Not refereed]
   

  Endothelin receptors (ETRs) is one of G protein coupled receptors, and consist of ET type A receptor (ET_AR) and ET type B receptor (ET_BR). 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.
• 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  [Not refereed]
   

  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
• 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)  2021
• The 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-  2021
• Annexin 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  [Not refereed]
   

  Endothelin receptors (ETRs) is one of G protein coupled receptors, and consist of ET type A receptor (ET_AR) and ET type B receptor (ET_BR). 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 ET_AR and ET_BR. 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.
• Ca²⁺-sensing receptor-G_q/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  [Not refereed]
   

  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 G_q/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 G_q/11 protein-eNOS-dependent pathway.
• Molecular 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  [Not refereed]
   

  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.
• 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-  2020
• GRP78 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  [Not refereed]
   

  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 (ET_AR) and ET type B receptor (ET_BR). ET_AR and ET_BR generally exhibit the opposite responses, although many exceptions exist. Here, we attempted to identify ET_AR or ET_BR specific binding proteins to understand difference of ET_AR- and ET_BR-mediated responses upon ET-1 stimulation. We found that GRP78 exhibited a stronger binding affinity toward ET_BR than ET_AR. Overexpression of GRP78 promotes ET_BR-mediated ERK activation. In addition, the silencing of GRP78 suppressed ET_BR-mediated ERK activation. On the other hand, ET_BR can localize GRP78 to cell periphery. Our results suggest that interaction of ET_BR with GRP78 affects the ERK activation and GRP78 localization.
• Elucidation 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  [Not refereed]
   

  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.
• Involvement 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  [Not refereed]
   

  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 G_q/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 G_q/11-protein-dependent activation of eNOS.
• 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
• 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)  2019
• MFN2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  2018/04  

  インスリン抵抗性とは、骨格筋、脂肪細胞、肝臓などで、インスリンに対する感受性が低下した病態を指す。インスリン抵抗性では、インスリンによる正常なグルコース(糖)代謝が障害されているため、2型糖尿病の発症につながる。近年、社会の高齢化や都市化に伴う食生活の変化や運動量の減少によって、2型糖尿病患者が急増していることから、インスリン抵抗性の病態基盤の解明は、焦眉の課題となっている。グルコースは、脳、骨格筋、脂肪、内臓などで代謝・利用されているが、全身のグルコース利用に対する各組織の寄与率は大きく異なる。健常者の骨格筋におけるグルコース利用率は、全身の70%を占めているが、2型糖尿病患者では、骨格筋におけるグルコース利用率が特異的に半減している。このことは、骨格筋におけるインスリン抵抗性が2型糖尿病の発症に関与している可能性を示唆している。血管収縮性・炎症性ペプチドであるエンドセリン-1(ET-1)は、インスリン抵抗性を惹起することが報告されているが、その詳細な発症機序は不明である。本研究では、ラットL6筋管細胞を用いて、インスリンシグナルに対するET-1の作用を薬理学的に解析した。L6筋管細胞において、インスリンは、PI3キナーゼの活性化を介して、Aktのリン酸化とグルコースの取り込みを促進した。ET-1は、インスリンによるAktリン酸化とグルコース取り込みを抑制し、このET-1の抑制作用は、選択的エンドセリンA型受容体(ETA R)遮断薬や選択的Gq/11タンパク質阻害薬の前処理及びsiRNAによる内在性Gタンパク質共役型受容体キナーゼ2(GRK2)の発現抑制によって解除された。また、ET-1は、AktとGRK2との相互作用を増強した。以上の結果から、L6筋管細胞において、ETA R及びGRK2が、ET-1によるインスリンシグナルの抑制に関与していると考えられた。(著者抄録)
• 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
• 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
• 不飽和カルボニル化合物による細胞傷害機構の解明

  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  69th-  250  -250  2017
• 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
• 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  [Not refereed][Not invited]
  
• Phos-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  2016/08
• 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  2016/05
• 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  

  肺動脈性肺高血圧症(pulmonary arterial hypertension:PAH)は、肺血管の拡張能低下や肺血管床の器質的変化(肺血管リモデリング)を病態基盤とする難治性呼吸器疾患である。エンドセリン-1(ET-1)は、血管内皮細胞で産生される血管収縮性・炎症性ペプチドであり、血管収縮・炎症・線維化・細胞増殖・アポトーシス抵抗性獲得などを引き起こすことによって、肺血管リモデリングを促進させ、PAHの病態を悪化させる。PAHの発症・進展には、血管内皮細胞の機能不全による内皮由来弛緩因子(プロスタサイクリン(PGI2)と一酸化窒素(NO))の産生低下も関与している。そのため、PAHの肺血管拡張療法では、血管平滑筋に対する内皮由来弛緩因子と内皮由来収縮因子の作用の不均衡を是正するために、プロスタサイクリン経路を活性化するプロスタノイドIP受容体(PGI2受容体)活性薬、NO経路を活性化する可溶性グアニル酸シクラーゼ刺激薬やホスホジエステラーゼ5型阻害薬、ならびに、ET経路を抑制するET受容体(ETR)遮断薬が用いられている。最近では、PAHの病態解明が進み、ETRシグナルの下流で活性化されるTRPC6チャネルやRhoキナーゼが、血管平滑筋細胞の収縮や増殖を介して、PAHの病態形成に関与していることが明らかにされている。本稿では、本邦において承認されているPAH治療薬の作用機序や臨床エビデンスを概説した後、エンドセリンシステムに対するPAH治療薬の作用、ならびに、今後のPAH治療戦略について考察する。(著者抄録)
• 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)  2016
• Unsaturated 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  [Not refereed][Not invited]
  
• 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  2015/09  

  肺血管リモデリングは、肺動脈内皮細胞・平滑筋細胞の異常増殖とアポトーシス耐性獲得によって惹起される器質的な肺血管病変であり、肺動脈性肺高血圧症(pulmonary arterial hypertension:PAH)の発症・進展に深く関与している。エンドセリン-1(endothelin-1:ET-1)は、血管内皮細胞で産生される血管収縮性・炎症性ペプチドであり、PAHの病態形成において、重要な役割を担っている。ET-1によるエンドセリンA型受容体刺激は、transient receptor potential canonical 6(TRPC6)チャネルの活性化に伴う持続的なCa2+流入やRhoキナーゼの活性化を介して、肺血管リモデリングを引き起こす。一方、ET-1によるエンドセリンB型受容体の活性化は、血管内皮細胞における内皮由来弛緩因子の遊離および血中ET-1のクリアランスを介して、肺血管リモデリングの抑制に寄与する。本稿では、エンドセリン受容体シグナルに焦点を当て、肺血管リモデリングの発症機序と治療戦略について概説する。(著者抄録)
• New 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  2015/09
• 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  2011/05  [Not refereed][Not invited]
  
• 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  2010/05  [Not refereed][Not invited]
  
• 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  2009/05  [Not refereed][Not invited]
  
• 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  2008/06  [Not refereed][Not invited]
  
• 好中球の化学走性におけるArf GEFの役割

  真崎雄一, 佐邊壽孝  生化学  80回・30回-  2P-0421  -15  2007/11  [Not refereed][Not invited]
  
• Fbx8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  2007/11  [Not refereed][Not invited]
  
• Arf6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  2007/08  [Not refereed][Not invited]
  
• [Directional sensing and superoxide production in neutrophils].

  Yuichi Mazaki  Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme  51-  (6 Suppl)  727  -32  2006/05  [Refereed][Not invited]
  
• 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  2004/09  [Not refereed][Not invited]
  
• Roles 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  2004/05  [Not refereed][Not invited]
  
• 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  2003/08  [Not refereed][Not invited]
  
• 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  2002/05  [Not refereed][Not invited]
  
• 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  2001/05  [Not refereed][Not invited]
  
• Interaction of paxillin with p21-activated kinase (PAK)

  S Hashimoto, A Tsubouchi, Y Mazaki, H Sabe  MOLECULAR BIOLOGY OF THE CELL  11-  172A  -173A  2000/12  [Not refereed][Not invited]
  
• 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  2000/08  [Not refereed][Not invited]
  
• ARF 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  1999/08  [Not refereed][Not invited]
  
• ARF 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  1999/08  [Not refereed][Not invited]
  
• ARF 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-  1999
• ARF6 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-  1999
• Paxillin 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  1998/12/01  [Not refereed][Not invited]
  
• A 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  1998/12/01  [Not refereed][Not invited]
  
• Analysis 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  1998/12/01  [Not refereed][Not invited]
  
• Epithelial 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  1998/12/01  [Not refereed][Not invited]
  
• 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  1998/08  [Not refereed][Not invited]
  
• 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  1998/08  [Not refereed][Not invited]
  
• 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  1998/08  [Not refereed][Not invited]
  
• 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  1998/08  [Not refereed][Not invited]
  
• 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  1997/05
• Analysis 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-  1997
• Analysis 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-  1996
• The 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-  1995
• Expression 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-  1994

Books etc

• 細胞骨格と細胞運動

  (Joint workFAKとパキシリン)
  シュプリンガー・フェアラーク東京 2002

Association Memberships

• THE JAPANESE PHARMACOLOGICAL SOCIETY   THE BIOPHYSICAL SOCIETY OF JAPAN   THE MOLECULAR BIOLOGY SOCIETY OF JAPAN   

Research Projects

• 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

  日本学術振興会：科学研究費助成事業

  Date (from‐to) : 2021/04 -2024/03 

  Author : 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。
• Elucidation of the mechanisms of neutrophil mitochondrial fusion in infection

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2018/04 -2021/03 

  Author : 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.
• Roles of ARFs in superoxide production and chemotaxis of neutrophil

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2013/04 -2016/03 

  Author : 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.
• Roles of GBF1 in cell migration of neutrophil

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2009 -2011 

  Author : 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.
• Roles of ARF in polarity formation during cell migration

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2007 -2008 

  Author : 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。
• Roles played by Arf6 in cell migration and invasion

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2004 -2005 

  Author : 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.
• 新規パキシリン結合タンパク質PAG1の細胞運動における役割

  日本学術振興会：科学研究費助成事業

  Date (from‐to) : 2001 -2001 

  Author : 真崎 雄一
• Role of paxillin-associatcd ARFGAPs in cell migration.

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2000 -2001 

  Author : 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.
• 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

  日本学術振興会：科学研究費助成事業

  Date (from‐to) : 2000 -2001 

  Author : 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。
• 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

  日本学術振興会：科学研究費助成事業

  Date (from‐to) : 1999 -2001 

  Author : 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。
• 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

  日本学術振興会：科学研究費助成事業

  Date (from‐to) : 2000 -2000 

  Author : 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。
• 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

  日本学術振興会：科学研究費助成事業

  Date (from‐to) : 1999 -2000 

  Author : 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。
• 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

  日本学術振興会：科学研究費助成事業

  Date (from‐to) : 1999 -1999 

  Author : 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。
• Role of paxillin isoforms and their tyrosine phosphorylation in cell migration.

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 1998 -1999 

  Author : 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.
• 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

  日本学術振興会：科学研究費助成事業

  Date (from‐to) : 1998 -1998 

  Author : 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。