基本情報

所属

• 医学研究院　生理系部門　薬理学分野

職名

• 講師

学位

• 博士（理学）（総合研究大学院大学）

ホームページURL

http://plaza.umin.ac.jp/cellular_pharm/

科研費研究者番号

• 60311304

J-Global ID

• 200901032767342966

研究キーワード

• 好中球   ミトコンドリア   細胞運動   膜輸送   細胞極性   

研究分野

• ライフサイエンス / 医化学
• ライフサイエンス / 細胞生物学
• ライフサイエンス / 薬理学

職歴

• 2015年 - 現在 北海道大学 医学研究科 講師
• 2013年 - 2015年 産業医科大学 医学部 講師
• 2008年 - 2013年 熊本大学 大学院先導機構 特任助教
• 2007年 - 2008年 大阪バイオサイエンス研究所 第一研究部 研究員

所属学協会

• 日本薬理学会   日本生物物理学会   日本分子生物学会   

研究活動情報

論文

• 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年09月01日 [査読有り][通常論文]
   

  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年08月 [査読有り][通常論文]
   

  This study examined the possible involvement of Ca2+-sensing receptor (CaSR) in nitric oxide (NO) production in human vascular endothelial cells. Extracellular Ca2+ elevated the intracellular Ca2+ concentration, the endothelial NO synthase (eNOS) phosphorylation level, and NO release from the cells. These responses were inhibited by a CaSR antagonist and a Gq/11 protein inhibitor. Application of an endothelial cell suspension induced vasorelaxation in isolated rat thoracic aorta precontracted by phenylephrine. Adding an NO scavenger to the organ bath abolished this vasorelaxation response. These results suggest that extracellular Ca2+ promotes NO generation via CaSR- and Gq/11 protein-mediated eNOS activation.
• Mitofusin 2 is involved in chemotaxis of neutrophil-like differentiated HL-60 cells

  Yuichi Mazaki, Shingo Takada, Junko Nio-Kobayashi, Satoshi Maekawa, Tsunehito Higashi, Yasuhito Onodera, Hisataka Sabe

  Biochemical and Biophysical Research Communications 513 3 708 - 713 Elsevier {BV} 2019年06月04日 [査読有り][通常論文]
   

  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年05月 [査読有り][通常論文]
   

  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 2019年03月26日 [査読有り][通常論文]
   

  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 2019年03月 [査読有り][通常論文]
   

  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 2019年02月19日 [査読有り][通常論文]
   

  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年 [査読有り][通常論文]
   

  A recent case report described a case of pulmonary arterial hypertension (PAH) associated with use of the Chinese herbal medicine Qing-Dai; however, the clinical course and possible mechanisms have not been characterized. We present the case of a man with ulcerative colitis who was diagnosed with idiopathic PAH. After initiating oral beraprost therapy, the patient showed significant hemodynamic improvements and an unusual course of clinical recovery. In 2016, the Japanese Ministry of Health, Labour, and Welfare issued a warning regarding the possible side effects of Qing-Dai. We learned that our patient had been taking self-purchased Qing-Dai for 2 years. Therefore, we performed an experimental study and determined that Qing-Dai may cause PAH through a mechanism involving nitric oxide synthase inhibition and pulmonary artery endothelial dysfunction.
• Protein kinase C-dependent cell damage by unsaturated carbonyl compounds in vascular cells

  Tsunehito Higashi, Yosuke Mai, Yuichi Mazaki

  Journal of Bioscience and Bioengineering 126 4 527 - 532 Elsevier {BV} 2018年10月 [査読有り][通常論文]
   

  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 151 4 140 - 147 Japanese Pharmacological Society 2018年 [査読有り][招待有り]
  
• 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 2017年12月 [査読有り][通常論文]
   

  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 2017年10月02日 [査読有り][通常論文]
   

  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 2016年03月 [査読有り][通常論文]
   

  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 2016年 [査読有り][通常論文]
   

  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 2016年 [査読有り][通常論文]
   

  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  2016年 [査読有り][通常論文]
   

  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 2016年 [査読有り][通常論文]
   

  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.

  Mazaki Y, Nishimura Y, Sabe H

  Molecular biology of the cell 23 13 2457 - 67 2012年07月 [査読有り][通常論文]
   

  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 2008年03月 [査読有り][通常論文]
   

  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 2008年01月 [査読有り][通常論文]
   

  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 2007年02月07日 [査読有り][通常論文]
   

  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 2006年10月 [査読有り][通常論文]
   

  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 2006年07月 [査読有り][通常論文]
   

  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 2005年03月09日 [査読有り][通常論文]
   

  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 166 2 283 - 95 Rockefeller University Press 2004年07月19日 [査読有り][通常論文]
   

  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 2002年11月25日 [査読有り][通常論文]
   

  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 2002年02月22日 [査読有り][通常論文]
   

  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/Papα/Kiaa0400, a Gtpase-Activating Protein for Adp-Ribosylation Factor (Arf), Regulates Arf6 in Fcγ Receptor–Mediated Phagocytosis of Macrophages

  Hiroshi Uchida, Akiko Kondo, Yasunori Yoshimura, Yuichi Mazaki, Hisataka Sabe

  The Journal of Experimental Medicine 193 8 955 - 966 Rockefeller University Press 2001年04月 [査読有り][通常論文]
   

  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 2001年03月 [査読有り][通常論文]
   

  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 2001年02月23日 [査読有り][通常論文]
   

  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/Papα/KIAA0400, Bearing an ADP-Ribosylation Factor GTPase-activating Protein Activity, Is Involved in Paxillin Recruitment to Focal Adhesions and Cell Migration

  Akiko Kondo, Shigeru Hashimoto, Hajime Yano, Kuniaki Nagayama, Yuichi Mazaki, Hisataka Sabe

  Molecular Biology of the Cell 11 4 1315 - 1327 American Society for Cell Biology ({ASCB}) 2000年04月 [査読有り][通常論文]
   

  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 2000年02月 [査読有り][通常論文]
   

  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 1998年08月 [査読有り][通常論文]
   

  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 1998年01月 [査読有り][通常論文]
   

  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 1997年10月 [査読有り][通常論文]
   

  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.
• 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 (株)ニュー・サイエンス社 1997年05月
• 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 1997年03月 [査読有り][通常論文]
   

  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 1996年08月 [査読有り][通常論文]
   

  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.

書籍

• 細胞骨格と細胞運動

  (担当:共著範囲:FAKとパキシリン)
  シュプリンガー・フェアラーク東京 2002年

その他活動・業績

• 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年
• 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年
• 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年 [査読無し][通常論文]
  
• 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年 [査読無し][通常論文]
  
• エンドセリン受容体シグナルからの視点 (東邦医学会雑誌) -- (第146回東邦医学会例会 パネルディスカッション 肺血管リモデリングの発症機序に迫る)

  堀之内 孝広, 東 恒仁, 真崎 雄一 Toho journal of medicine 1 (3) 197 -199 2015年09月
• 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年05月 [査読無し][通常論文]
  
• 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年05月 [査読無し][通常論文]
  
• 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年05月 [査読無し][通常論文]
  
• 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年06月 [査読無し][通常論文]
  
• 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年11月 [査読無し][通常論文]
  
• 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年11月 [査読無し][通常論文]
  
• 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年08月 [査読無し][通常論文]
  
• 好中球の指向性センシングと活性酸素産生

  Yuichi Mazaki 蛋白質核酸酵素 51 (6 Suppl) 727 -32 2006年05月 [査読有り][通常論文]
  
• 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年09月 [査読無し][通常論文]
  
• 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年05月 [査読無し][通常論文]
  
• 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年08月 [査読無し][通常論文]
  
• 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年05月 [査読無し][通常論文]
  
• 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年05月 [査読無し][通常論文]
  
• 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年12月 [査読無し][通常論文]
  
• 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年08月 [査読無し][通常論文]
  
• 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年08月 [査読無し][通常論文]
  
• 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年08月 [査読無し][通常論文]
  
• 上皮間充織形質転換におけるパキシリンの機能動態

  内田 浩, 矢野 元, 真崎 雄一, 橋本 茂, 佐邊 壽孝 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年12月01日 [査読無し][通常論文]
  
• 接着斑タンパク質パキシリンの核周辺域への局在機構

  真崎 雄一, 矢野 元, 大川 克也, 橋本 茂, 岩松 明彦, 佐邊 壽孝 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年12月01日 [査読無し][通常論文]
  
• パキシリン結合性新規蛋白質の解析

  近藤 明子, 橋本 茂, 真崎 雄一, 永山 国昭, 佐邊 壽孝 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年12月01日 [査読無し][通常論文]
  
• 細胞接着を介する上皮細胞生存性シグナルの解析

  橋本 茂, 真崎 雄一, 内田 浩, 佐邊 壽孝 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年12月01日 [査読無し][通常論文]
  
• 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年08月 [査読無し][通常論文]
  
• 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年08月 [査読無し][通常論文]
  
• 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年08月 [査読無し][通常論文]
  
• 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年08月 [査読無し][通常論文]
  

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