Protein kinase Cβ is involved in cigarette smoke gas phase-induced ferroptosis in J774 macrophages
Tsunehito Higashi, Haruka Handa, Yosuke Mai, Katsumi Maenaka, Takashi Tadokoro
Journal of Pharmacological Sciences, 153, 1, 22, 25, Elsevier BV, 2023年09月, ［査読有り］, ［筆頭著者, 責任著者］
研究論文（学術雑誌）

タバコ煙ガス相はJ774マクロファージにおいてPKCβを介してフェロトーシスを誘導する
東 恒仁, 半田 悠, 眞井 洋輔, 前仲 勝実, 田所 高志
日本薬理学会年会要旨集, 97, 3-B-P-069, 公益社団法人 日本薬理学会, 2023年
日本語, Cigarette smoking is a risk factor for various types of diseases including atherosclerosis, hypertension, chronic obstructive pulmonary disease, and respiratory infection. The respiratory infection caused by cigarette smoking is due to immune cell dysfunction by cigarette smoke, although its molecular mechanism remains to be clarified. The cigarette smoke can be divided into two phases: tar (particle) phase and gas phase. We have previously reported that gas phase extract of cigarette smoke (CSE) induces cell death. In this study, we have examined the effects of CSE on J774 macrophages. CSE and unsaturated carbonyl compounds, cytotoxic factors in the CSE, induced cell death in J774 macrophages. Ferrostatin-1 and liproxstatin-1, ferroptosis inhibitors, suppressed cell death caused by CSE and unsaturated carbonyl compounds. A broad-range protein kinase C (PKC) inhibitor Gö6983 suppressed CSE- and unsaturated carbonyl compounds-induced cell death. To identify PKC isoforms involved in the process, we have examined isoform-specific inhibitors. Enzastaurin, a PKCβ-specific inhibitor, suppressed the cell death. Enzastaurin also suppressed RSL3-induced ferroptosis. These results suggest that CSE and unsaturated carbonyl compounds induce PKCβ-dependent ferroptosis in J774 macrophages.

Decreased proteasomal function induces neuronal loss and memory impairment
Tomaru U, Ito T, Ohmura Y, Higashikawa K, Miyajima S, Tomatsu R, Higashi T, Ishizu A, Kuge Y, Yoshioka M, Kasahara M
Am J Pathol, 191, 1, 144, 156, 2021年01月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Alzheimer disease (AD) is a progressive neurodegenerative disorder and the most common type of dementia worldwide. There is considerable evidence of age-related disruption of proteostasis being responsible for the development of AD. The proteasome is a multicatalytic enzyme complex that degrades both normal and damaged proteins, and an age-related decline in its activity has been implicated in age-related pathologies. Although proteasomal dysfunction is assumed to be a key AD hallmark, it remains unclear whether its role in disease onset is causative or secondary. In this study, we demonstrate that mice with proteasomal dysfunction exhibited memory impairment with associated neuronal loss, accumulation of phosphorylated tau, and activation of endoplasmic reticulum (ER) stress-related apoptosis pathways. Impaired proteasomal activity also activated ER stress-related apoptosis pathways in HT-22, a murine hippocampal neuronal cell line. HT-22 cell death, caused by proteasomal inhibition, was prevented by an inhibitor of c-Jun N-terminal kinase, an ER stress-related molecule. Collective evidence suggests that impaired proteasomal activity alters proteostasis, and subsequent ER stress-mediated pathways play pivotal roles in neuronal loss. Because aging decreases proteasomal function, age-related impairment of proteasomes may be involved in the development and progression of AD in elderly patients.

Cigarette smoke extract induces ferroptosis in vascular smooth muscle cells
Sampilvanjil A, Karasawa T, Yamada N, Komada T, Higashi T, Baatarjav C, Watanabe S, Kamata R, Ohno N, Takahashi M
Am J Physiol Heart Circ Physiol, 318, 3, H508, H518, 2020年03月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Cigarette smoking is a major risk factor for aortic aneurysm and dissection; however, no causative link between smoking and these aortic disorders has been proven. In the present study, we investigated the mechanism by which cigarette smoke affects vascular wall cells and found that cigarette smoke extract (CSE) induced a novel form of regulated cell death termed ferroptosis in vascular smooth muscle cells (VSMCs). CSE markedly induced cell death in A7r5 cells and primary rat VSMCs, but not in endothelial cells, which was completely inhibited by specific ferroptosis inhibitors [ferrostatin-1 (Fer-1) and Liproxstatin-1] and an iron chelator (deferoxamine). CSE-induced VSMC death was partially inhibited by a GSH precursor (N-acetyl cysteine) and an NADPH oxidase inhibitor [diphenyleneiodonium chloride (DPI)], but not by inhibitors of pan-caspases (Z-VAD), caspase-1 (Z-YVAD), or necroptosis (necrostatin-1). CSE also upregulated IL-1β, IL-6, TNF-α, matrix metalloproteinase (MMP)-2, MMP-9, and TIMP-1 (tissue inhibitor of metalloproteinase)in A7r5 cells, which was inhibited by Fer-1. Furthermore, CSE induced the upregulation of Ptgs2 mRNA, lipid peroxidation, and intracellular GSH depletion, which are key features of ferroptosis. VSMC ferroptosis was induced by acrolein and methyl vinyl ketone, major constituents of CSE. Furthermore, CSE caused medial VSMC loss in ex vivo aortas. Electron microscopy analysis showed mitochondrial damage and fragmentation in medial VSMCs of CSE-treated aortas. All of these manifestations were partially restored by Fer-1. These findings demonstrate that ferroptosis is responsible for CSE-induced VSMC death and suggest that ferroptosis is a potential therapeutic target for preventing aortic aneurysm and dissection.NEW & NOTEWORTHY Cigarette smoke extract (CSE)-induced cell death in rat vascular smooth muscle cells (VSMCs) was completely inhibited by specific ferroptosis inhibitors and an iron chelator. CSE also induced the upregulation of Ptgs2 mRNA, lipid peroxidation, and intracellular GSH depletion, which are key features of ferroptosis. CSE caused medial VSMC loss in ex vivo aortas. These findings demonstrate that ferroptosis is responsible for CSE-induced VSMC death.

Pyrenosine A induces monopolar spindle formation and suppress proliferation of cancer cells
Myobatake Y, Kamisuki S, Tsukuda S, Higashi T, Chinen T, Takemoto K, Hachisuka M, Suzuki Y, Takei M, Tsurukawa Y, Maekawa H, Takeuchi T, Matsunaga TM, Sahara H, Usui T, Matsunaga S, Sugawara F
Bioorg Med Chem, 27, 23, 115149, 115149, 2019年12月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Pyrenocine A, a phytotoxin, was found to exhibit cytotoxicity against cancer cells with an IC50 value of 2.6-12.9 μM. Live cell imaging analysis revealed that pyrenocine A arrested HeLa cells at the M phase with characteristic ring-shaped chromosomes. Furthermore, as a result of immunofluorescence staining analysis, we found that pyrenocine A resulted in the formation of monopolar spindles in HeLa cells. Monopolar spindles are known to be induced by inhibitors of the kinesin motor protein Eg5 such as monastrol and STLC. Monastrol and STLC induce monopolar spindle formation and M phase arrest via inhibition of the ATPase activity of Eg5. Interestingly, our data revealed that pyrenocine A had no effect on the ATPase activity of Eg5 in vitro, which suggested the compound induces a monopolar spindle by an unknown mechanism. Structure-activity relationship analysis indicates that the enone structure of pyrenocine A is likely to be important for its cytotoxicity. An alkyne-tagged analog of pyrenocine A was synthesized and suppressed proliferation of HeLa cells with an IC50 value of 2.3 μM. We concluded that pyrenocine A induced monopolar spindle formation by a novel mechanism other than direct inhibition of Eg5 motor activity, and the activity of pyrenocine A may suggest a new anticancer mechanism.

Mitofusin 2 is involved in chemotaxis of neutrophil-like differentiated HL-60 cells
Mazaki Y, Takada S, Nio-Kobayashi J, Maekawa S, Higashi T, Onodera Y, Sabe H
Biochem Biophys Res Commun, 513, 3, 708, 713, 2019年06月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Endothelin type B receptor interacts with 78-kDa glucose-regulated protein
Mazaki Y, Higashi T, Onodera Y, Nam JM, Hashimoto A, Hashimoto S, Horinouchi T, Miwa S
FEBS Lett, 593, 6, 644, 651, 2019年03月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Annexin A2 is involved in activation of extracellular signal-regulated kinase upon endothelin-1 stimulation
Mazaki Y, Higashi T, Horinouchi T, Miwa S
Biochem Biophys Res Commun, 511, 1, 69, 72, 2019年03月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Glutathione and cysteines suppress cytotoxicity of gas phase of cigarette smoke by direct reacting with unsaturated carbonyl compounds in the gas phase　　　　　　　　　　　　　　　
Higashi T, Elmeligy E, Mai Y, Noya Y, Terada K, Mazaki Y, Kuge Y, Miwa S
Biochem Biophys Res Commun, 509, 4, 988, 993, 2019年02月, ［査読有り］, ［筆頭著者, 責任著者］
英語, 研究論文（学術雑誌）

Autoantibodies undetectable by chemiluminescent enzyme immunoassay require extended antigen-antibody reaction time for detection
Mai Y, Ujiie H, Higashi T, Yamagami J, Iwata H, Shimizu H
Br J Dermatol, 180, 1, 215, 216, 2019年01月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）

CLEIAで検出できない抗デスモグレイン3抗体の特徴　　　　　　　　　　　　　　　
眞井 洋輔, 氏家 英之, 東 恒仁, 岩田 浩明, 清水 宏
日本皮膚科学会雑誌, 128, 5, 1201, 1201, (公社)日本皮膚科学会, 2018年05月
日本語

Protein kinase C-dependent cell damage by unsaturated carbonyl compounds in vascular cells
Tsunehito Higashi, Yosuke Mai, Yuichi Mazaki
Journal of Bioscience and Bioengineering, 126, 4, 527, 532, Elsevier B.V., 2018年, ［査読有り］, ［筆頭著者］
英語, 研究論文（学術雑誌）, 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.

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, SOC BIOSCIENCE BIOENGINEERING JAPAN, 2017年12月, ［査読有り］, ［筆頭著者, 責任著者］
英語, 研究論文（学術雑誌）, 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. (C) 2017, The Society for Biotechnology, Japan. All rights reserved.

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, 15, 36, BIOMED CENTRAL LTD, 2017年10月, ［査読有り］
英語, 研究論文（学術雑誌）, 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 G beta gamma.-PAK1-aPIX-GIT2 linear complex during GPCR-induced chemotaxis, in which aPIX 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, aPIX, 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.

Cigarette smoke extract inhibits platelet aggregation by suppressing cyclooxygenase activity
Kashiwagi H, Yuhki K, Imamichi Y, Kojima F, Kumei S, Higashi T, Horinouchi T, Miwa S, Narumiya S, Ushikubi F
TH Open, 1, 2, e122, e129, 2017年07月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, The results of studies that were performed to determine whether cigarette smoking affects platelet function have been controversial, and the effects of nicotine- and tar-free cigarette smoke extract (CSE) on platelet function remain to be determined. The aim of this study was to determine the effect of CSE on platelet aggregation and to clarify the mechanism by which CSE affects platelet function. CSE inhibited murine platelet aggregation induced by 9,11-dideoxy-9α,11α-methanoepoxy-prosta-5Z,13E-dien-1-oic acid (U-46619), a thromboxane (TX) A 2 receptor agonist, and that induced by collagen with respective IC 50 values of 1.05 ± 0.14% and 1.34 ± 0.19%. A similar inhibitory action of CSE was also observed in human platelets. CSE inhibited arachidonic acid-induced TXA 2 production in murine platelets with an IC 50 value of 7.32 ± 2.00%. Accordingly, the inhibitory effect of CSE on collagen-induced aggregation was significantly blunted in platelets lacking the TXA 2 receptor compared with the inhibitory effect in control platelets. In contrast, the antiplatelet effects of CSE in platelets lacking each inhibitory prostanoid receptor, prostaglandin (PG) I 2 receptor and PGE 2 receptor subtypes EP 2 and EP 4 , were not significantly different from the effects in respective control platelets. Among the enzymes responsible for TXA 2 production in platelets, the activity of cyclooxygenase (COX)-1 was inhibited by CSE with an IC 50 value of 1.07 ± 0.15% in an uncompetitive manner. In contrast, the activity of TX synthase was enhanced by CSE. The results indicate that CSE inhibits COX-1 activity and thereby decreases TXA 2 production in platelets, leading to inhibition of platelet aggregation.

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, Tsunehito Higashi, Toshiyuki Fukazawa, Hiroyoshi Fujita, Hidenao Sasaki, Hiroshi Wakao
SPRINGERPLUS, 5, 1, 1, 16, SPRINGER INTERNATIONAL PUBLISHING AG, 2016年08月, ［査読有り］
英語, 研究論文（学術雑誌）, 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 CD8high 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-alpha and IFN-gamma 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.

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, PHARMACEUTICAL SOC JAPAN, 2016年06月, ［査読有り］, ［招待有り］, ［筆頭著者, 責任著者］
英語, 研究論文（学術雑誌）, 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 1.050L/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 mu m) 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.

骨格筋細胞においてエンドセリン-1はGタンパク質共役型受容体キナーゼ2を介してインスリン抵抗性を惹起する　　　　　　　　　　　　　　　
堀之内孝広, 星暁壮, 原田拓弥, 比嘉綱己, サリタ・カルキ, 寺田晃士, 東恒仁, 眞井洋輔, ネパル・プラハ, 真崎雄一, 三輪聡一
北海道医学雑誌, 91, 77, 2016年
日本語, 研究論文（学術雑誌）

Endothelin-1 suppresses insulin-stimulated Akt phosphorylation and glucose uptake via G protein-coupled receptor kinase 2 in skeletal muscle cells
Horinouchi T, Hoshi A, Harada T, Higa T, Karki S, Terada K, Higashi T, Mai Y, Nepal P, Mazaki Y, Miwa S
Br. J. Pharmacol., 173, 6, 1018, 1032, 2016年, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

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年, ［査読有り］, ［招待有り］, ［国内誌］
日本語, 研究論文（学術雑誌）

Carbonyl compounds in the gas phase of cigarette mainstream smoke and their pharmacological properties.
Horinouchi T, Higashi T, Mazaki Y, Miwa S
Biol. Pharm. Bull., 39, 6, 909, 914, 2016年, ［査読有り］, ［招待有り］, ［国内誌］
英語, 研究論文（学術雑誌）, 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.

Using Phos-tag in Western blotting analysis to evaluate protein phosphorylation.
Horinouchi T, Terada K, Higashi T, Miwa S
Methods in Molecular Biology., 1397, 266, 277, 2016年, ［査読有り］, ［招待有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Protein phosphorylation has traditionally been detected by radioisotope phosphate labeling of proteins with radioactive ATP. Several nonradioactive assays with phosphorylation site-specific antibodies are now available for the analysis of phosphorylation status at target sites. However, due to their high specificity, these antibodies they cannot be used to detect unidentified phosphorylation sites. Recently, Phos-tag technology has been developed to overcome the disadvantages and limitations of phosphospecific antibodies. Phos-tag and its derivatives conjugated to biotin, acrylamide, or agarose, form alkoxide-bridged dinuclear metal complexes, which can capture phosphate monoester dianions bound to serine, threonine, and tyrosine residues, in an amino acid sequence-independent manner. Here, we describe our method, which is based on in vitro kinase assay and Western blotting analysis using biotinylated Phos-tag and horseradish peroxidase-conjugated streptavidin, to determine the sites of TRPC6 (transient receptor potential canonical 6) channel phosphorylated by protein kinase A.

Decreased proteasomal function accelerates cigarette smoke-induced pulmonary emphysema in mice
Yosuke Yamada, Utano Tomaru, Akihiro Ishizu, Tomoki Ito, Takayuki Kiuchi, Ayako Ono, Syota Miyajima, Katsura Nagai, Tsunehito Higashi, Yoshihiro Matsuno, Hirotoshi Dosaka-Akita, Masaharu Nishimura, Soichi Miwa, Masanori Kasahara
LABORATORY INVESTIGATION, 95, 6, 625, 634, NATURE PUBLISHING GROUP, 2015年06月, ［査読有り］
英語, 研究論文（学術雑誌）, Chronic obstructive pulmonary disease (COPD) is a disease common in elderly people, characterized by progressive destruction of lung parenchyma and chronic inflammation of the airways. The pathogenesis of COPD remains unclear, but recent studies suggest that oxidative stress-induced apoptosis in alveolar cells contributes to emphysematous lung destruction. The proteasome is a multicatalytic enzyme complex that plays a critical role in proteostasis by rapidly destroying misfolded and modified proteins generated by oxidative and other stresses. Proteasome activity decreases with aging in many organs including lungs, and an age-related decline in proteasomal function has been implicated in various age-related pathologies. However, the role of the proteasome system in the pathogenesis of COPD has not been investigated. Recently, we have established a transgenic (Tg) mouse model with decreased proteasomal chymotrypsin-like activity, showing age-related phenotypes. Using this model, we demonstrate here that decreased proteasomal function accelerates cigarette smoke (CS)-induced pulmonary emphysema. CS-exposed Tg mice showed remarkable airspace enlargement and increased foci of inflammation compared with wild-type controls. Importantly, apoptotic cells were found in the alveolar walls of the affected lungs. Impaired proteasomal activity also enhanced apoptosis in cigarette smoke extract (CSE)-exposed fibroblastic cells derived from mice and humans in vitro. Notably, aggresome formation and prominent nuclear translocation of apoptosis-inducing factor were observed in CSE-exposed fibroblastic cells isolated from Tg mice. Collective evidence suggests that CS exposure and impaired proteasomal activity coordinately enhance apoptotic cell death in the alveolar walls that may be involved in the development and progression of emphysema in susceptible individuals such as the elderly.

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
東邦医学誌, 62, 3, 197, 199, The Medical Society of Toho University, 2015年, ［招待有り］
英語, 研究論文（学術雑誌）

Ubiquitination-regulated receptor trafficking of endothelin type A and type B receptors.
Terada K, Horinouchi T, Higashi T, Nepal P, Miwa S
Nihon yakurigaku zasshi. Folia pharmacologica Japonica, 145, 1, 4, 9, 2015年, ［査読有り］, ［国内誌］
日本語, 研究論文（学術雑誌）

Agonist-promoted Ubiquitination Differentially Regulates Receptor Trafficking of Endothelin Type A and Type B Receptors
Koji Terada, Takahiro Horinouchi, Yoichiro Fujioka, Tsunehito Higashi, Prabha Nepal, Mika Horiguchi, Sarita Karki, Chizuru Hatate, Akimasa Hoshi, Takuya Harada, Yosuke Mai, Yusuke Ohba, Soichi Miwa
JOURNAL OF BIOLOGICAL CHEMISTRY, 289, 51, 35283, 35295, AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, 2014年12月, ［査読有り］
英語, 研究論文（学術雑誌）, Two types of G protein-coupled receptors for endothelin-1 (ET-1), ET type A receptor (ETAR) and ETBR, closely resemble each other, but upon ET-1 stimulation, they follow totally different intracellular trafficking pathways; ETAR is recycled back to plasma membrane, whereas ETBR is targeted to lysosome for degradation. However, the mechanisms for such different fates are unknown. Here we demonstrated that ETBR but not ETAR was ubiquitinated on the cell surface following ET-1 stimulation and that ETBR was internalized and degraded in lysosome more rapidly than ETAR. The mutant ETBR (designated 5KR mutant) in which 5 lysine residues in the C-tail were substituted to arginine was not ubiquitinated, and its rates of internalization and degradation after ET-1 stimulation became slower, being comparable with those of ETAR. Confocal microscopic study showed that following ET-1 stimulation, ETAR and 5KR mutant of ETBR were co-localized mainly with Rab11, a marker of recycling endosome, whereas ETBR was co-localized with Rab7, a marker of late endosome/lysosome. In the 5KR mutant, ET-1-induced ERK phosphorylation and an increase in the intracellular Ca2+ concentration upon repetitive ET-1 stimulation were larger. A series of ETBR mutants (designated 4KR mutant), in which either one of 5 arginine residues of the 5KR mutant was reverted to lysine, were normally ubiquitinated, internalized, and degraded, with ERK phosphorylation being normalized. These results demonstrate that agonist-induced ubiquitination at either lysine residue in the C-tail of ETBR but not ETAR switches intracellular trafficking from recycling to plasma membrane to targeting to lysosome, causing decreases in the cell surface level of ETBR and intracellular signaling.

A Simple and Rapid Method for Standard Preparation of Gas Phase Extract of Cigarette Smoke
Tsunehito Higashi, Yosuke Mai, Yoichi Noya, Takahiro Horinouchi, Koji Terada, Akimasa Hoshi, Prabha Nepal, Takuya Harada, Mika Horiguchi, Chizuru Hatate, Yuji Kuge, Soichi Miwa
PLOS ONE, 9, 9, e107856, PUBLIC LIBRARY SCIENCE, 2014年09月, ［査読有り］, ［筆頭著者］
英語, 研究論文（学術雑誌）, Cigarette smoke consists of tar and gas phase: the latter is toxicologically important because it can pass through lung alveolar epithelium to enter the circulation. Here we attempt to establish a standard method for preparation of gas phase extract of cigarette smoke (CSE). CSE was prepared by continuously sucking cigarette smoke through a Cambridge filter to remove tar, followed by bubbling it into phosphate-buffered saline (PBS). An increase in dry weight of the filter was defined as tar weight. Characteristically, concentrations of CSEs were represented as virtual tar concentrations, assuming that tar on the filter was dissolved in PBS. CSEs prepared from smaller numbers of cigarettes (original tar concentrations <= 15 mg/ml) showed similar concentration-response curves for cytotoxicity versus virtual tar concentrations, but with CSEs from larger numbers (tar >= 20 mg/ml), the curves were shifted rightward. Accordingly, the cytotoxic activity was detected in PBS of the second reservoir downstream of the first one with larger numbers of cigarettes. CSEs prepared from various cigarette brands showed comparable concentration-response curves for cytotoxicity. Two types of CSEs prepared by continuous and puff smoking protocols were similar regarding concentration-response curves for cytotoxicity, pharmacology of their cytotoxicity, and concentrations of cytotoxic compounds. These data show that concentrations of CSEs expressed by virtual tar concentrations can be a reference value to normalize their cytotoxicity, irrespective of numbers of combusted cigarettes, cigarette brands and smoking protocols, if original tar concentrations are <= 15 mg/ml.

Endothelin-1 activates extracellular signal-regulated kinases 1/2 via transactivation of platelet-derived growth factor receptor in rat L6 myoblasts
Takuya Harada, Takahiro Horinouchi, Tsunaki Higa, Akimasa Hoshi, Tsunehito Higashi, Koji Terada, Yosuke Mai, Prabha Nepal, Mika Horiguchi, Chizuru Hatate, Soichi Miwa
LIFE SCIENCES, 104, 1-2, 24, 31, PERGAMON-ELSEVIER SCIENCE LTD, 2014年05月, ［査読有り］
英語, 研究論文（学術雑誌）, Aims: Endothelin (ET) system plays a critical role in the development of insulin resistance and type 2 diabetes. In skeletal muscle, differentiation of myoblasts to myotubes is accompanied by the development of insulin sensitivity. Activation of extracellular signal-regulated kinase (ERK) 1/2 inhibits the differentiation of myoblasts, leading to insulin resistance. Although ET receptor (ETR) stimulation generally activates ERK1/2, the mechanism for ETR-mediated ERK1/2 activation in skeletal muscle is unknown. The purpose of this study was to determine the signal transduction pathway involved in ET-1-stimulated ERK1/2 phosphorylation in L6 myoblasts derived from rat skeletal muscle.
Main methods: Changes in phosphorylation levels of ERK1/2 following stimulation with ET-1 were analyzed by Western blot in L6 myoblasts. To inhibit receptor internalization, dominant-negative dynamin (K44A) Was overexpressed in L6 myoblasts using adenovirus-mediated gene transfer.
Key findings: ET-1 induced phosphorylation of ERK1/2 in L6 myoblasts. The ERK1/2 phosphorylation was abolished by BQ123 (a selective ET type A receptor (ETAR) antagonist), YM-254890 (a G(alpha q/11) protein inhibitor), and AG370 (a platelet-derived growth factor receptor (PDGFR) kinase inhibitor), while U-73122 (a phospholipase C (PLC) inhibitor) was less potent. The ERK1/2 phosphorylation was inhibited by overexpression of dominant-negative dynamin (K44A). These results suggest that ETAR stimulation induces ERK1/2 phosphorylation in L6 myoblasts through G(q/11) protein-dependent, PLC-independent PDGFR transactivation which requires dynamin-dependent ETAR internalization.
Significance: Because activation of ERK1/2 is considered to inhibit differentiation of myoblasts with the development of insulin sensitivity, the ETAR-mediated PDGFR transactivation and subsequent ERK1/2 activation play an important role in ET-1-induced insulin resistance. (C) 2014 Elsevier Inc. All rights reserved.

Identification of stable cytotoxic factors in the gas phase extract of cigarette smoke and pharmacological characterization of their cytotoxicity
Yoichi Noya, Koh-ichi Seki, Hiroshi Asano, Yosuke Mai, Takahiro Horinouchi, Tsunehito Higashi, Koji Terada, Chizuru Hatate, Akimasa Hoshi, Prabha Nepal, Mika Horiguchi, Yuji Kuge, Soichi Miwa
Toxicology, 314, 1, 1, 10, Elsevier Ireland Ltd, 2013年12月06日, ［査読有り］
英語, 研究論文（学術雑誌）, Smoking is a major risk factor for atherosclerotic vascular diseases, but the mechanism for its genesis is unknown. We have recently shown that the gas phase of cigarette smoke (nicotine- and tar-free cigarette smoke extract
CSE) likely to reach the systemic circulation contains stable substances which cause cytotoxicity like plasma membrane damage and cell death in cultured cells, and also that the plasma membrane damage is caused through sequential activation of protein kinase C (PKC) and NADPH oxidase (NOX) and the resulting generation of reactive oxygen species (PKC/NOX-dependent mechanism), whereas cell death is caused through PKC/NOX-dependent and -independent mechanisms. To identify these stable substances, the CSE was prepared by passing the main-stream smoke of 10 cigarettes through a Cambridge glass fiber filter, trapping of the smoke in a vessel cooled at -80°C, and subsequent dissolution in 10ml of water. The CSE was fractionated into nine fractions using reversed-phase HPLC, and each fraction was screened for cytotoxicity in cultured cells, using propidium iodide uptake assay for cell membrane damage and MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] reduction assay for cell viability. The cytotoxicity was positive in two of the nine fractions (Fr2 and Fr5). After extraction of the active fractions into dichloromethane, GC/MS analysis identified 2-cyclopenten-1-one (CPO) in Fr5 but none in Fr2. After derivatization of the active fractions with O-(2,3,4,5,6-pentafluorobenzyl) hydroxylamine hydrochloride, GC/MS analysis identified acrolein, acetone and propionaldehyde in Fr2, and methyl vinyl ketone (MVK) in Fr5. After 4-h incubation, authentic acrolein and MVK induced concentration-dependent cytotoxicity with EC50 values of 75.9±8.2 and 47.0±8.0μM (mean±SEM
n=3), respectively, whereas acetone, propionaldehyde and CPO were without effect. However, after 24-h incubation, CPO induced concentration-dependent cytotoxicity with an EC50 value of 264.0±16.9μM (n=3). The concentrations of acrolein, MVK and CPO in the CSE were 3368±334, 2429±123 and 392.9±31.8μM (n=4), respectively, which were higher than the cytotoxic concentrations. The cytotoxicity of acrolein and MVK consisted of plasma membrane damage and decreased cell viability: the plasma membrane damage was totally prevented by treatment with an inhibitor of PKC or NOX, whereas the decreased cell viability was only partially prevented by these inhibitors. The cytotoxicity of CPO consisted only of decreased cell viability, which was totally resistant to these inhibitors. These results show that acrolein and MVK are responsible for the acute cytotoxicity of the CSE through PKC/NOX-dependent and -independent mechanisms, whereas CPO is responsible for the delayed cytotoxicity of the CSE through a PKC/NOX-independent mechanism. © 2013 Elsevier Ireland Ltd.

Application of visualization techniques for cell and tissue engineering
Tsunehito Higashi, Wataru Watanabe, Sachihiro Matsunaga
JOURNAL OF BIOSCIENCE AND BIOENGINEERING, 115, 2, 122, 126, SOC BIOSCIENCE BIOENGINEERING JAPAN, 2013年02月, ［査読有り］, ［招待有り］, ［筆頭著者, 責任著者］
英語, Visualization has been an indispensable technique in the biological field. The advantage of visualization is to perform non-disruptive analyses with high spatio and temporal resolution. Using these properties, visualization has been employed for cell and tissue engineering research, including therapeutic protein production, cell and organelle manipulation, and stem cell technology. For cell assessment and manipulation, two-photon microscopy based on femtosecond laser is becoming a major tool because of its high depth resolution, low cell damages, and depth of penetration into tick specimen. Non-disruptive and single cell observation/manipulation technique is a powerful tool for stem cell research. In this review, we discuss recent developments in cell and tissue engineering in relation to the revolution in visualization techniques. (C) 2012, The Society for Biotechnology, Japan. All rights reserved.

SON Protein Regulates GATA-2 through Transcriptional Control of the MicroRNA 23a similar to 27a similar to 24-2 Cluster
Erin Eun-Young Ahn, Tsunehito Higashi, Ming Yan, Shinobu Matsuura, Christopher J. Hickey, Miao-Chia Lo, Wei-Jong Shia, Russell C. DeKelver, Dong-Er Zhang
JOURNAL OF BIOLOGICAL CHEMISTRY, 288, 8, 5381, 5388, AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, 2013年02月, ［査読有り］
英語, 研究論文（学術雑誌）, SON is a DNA-and RNA-binding protein localized in nuclear speckles. Although its function in RNA splicing for effective cell cycle progression and genome stability was recently unveiled, other mechanisms of SON functions remain unexplored. Here, we report that SON regulates GATA-2, a key transcription factor involved in hematopoietic stem cell maintenance and differentiation. SON is highly expressed in undifferentiated hematopoietic stem/progenitor cells and leukemic blasts. SON knockdown leads to significant depletion of GATA-2 protein with marginal down-regulation of GATA-2 mRNA. We show that miR-27a is up-regulated upon SON knockdown and targets the 3'-UTR of GATA-2 mRNA in hematopoietic cells. Up-regulation of miR-27a was due to activation of the promoter of the miR-23a similar to 27a similar to 24-2 cluster, suggesting that SON suppresses this promoter to lower the microRNAs from this cluster. Our data revealed a previously unidentified role of SON in microRNA production via regulating the transcription process, thereby modulating GATA-2 at the protein level during hematopoietic differentiation.

Endothelin receptor signaling: new insight into its regulatory mechanisms.
Horinouchi T, Terada K, Higashi T, Miwa S
Journal of pharmacological sciences, 123, 2, 85, 101, 2, 2013年, ［査読有り］, ［国内誌］
英語, 研究論文（学術雑誌）, The endothelin (ET) system consists of two G protein coupled-receptors (GPCRs), ET type A receptor (ETAR) and ET type B receptor (ETBR), and three endogenous ligands, ET-1, ET-2, and ET-3. Stimulation of ETRs with ET-1 induces an increase in intracellular Ca(2+) concentration that is involved in a diverse array of physiological and pathophysiological processes, including vasoconstriction, and cell proliferation. Store-operated Ca(2+) entry and receptor-operated Ca(2+) entry triggered by activation of ETRs are regulated or modulated by endoplasmic reticulum Ca(2+) sensor (stromal interaction molecule 1) and voltage-independent cation channels (transient receptor potential canonical channels and Orai1). The ET-1-induced Ca(2+) mobilization results from activation of heterotrimeric G proteins by ETRs. In contrast, GPCR biology including modulation of receptor function and trafficking is regulated by a variety of GPCR interacting proteins (GIPs) that generally interact with the C-terminal domain of GPCRs. The ETR signaling is also regulated by GIPs such as Jun activation domain-binding protein 1. This review focuses on the regulatory mechanisms of the ETR signaling with special attention to the components involved in Ca(2+) signaling and to GIPs in the signal transduction, modification, and degradation of ETRs.

Nicotine- and Tar-free Cigarette Smoke Extract Induces Cell Injury via Intracellular Ca2+-Dependent Subtype-Specific Protein Kinase C Activation
Yosuke Mai, Tsunehito Higashi, Koji Terada, Chizuru Hatate, Prabha Nepal, Mika Horiguchi, Takuya Harada, Soichi Miwa, Takahiro Horinouchi
JOURNAL OF PHARMACOLOGICAL SCIENCES, 120, 4, 310, 314, JAPANESE PHARMACOLOGICAL SOC, 2012年12月, ［査読有り］
英語, 研究論文（学術雑誌）, Nicotine- and tar-free cigarette smoke extract (CSE) is reported to induce cell damage via activation of protein kinase C (PKC) and NADPH oxidase (NOX) in rat C6 glioma cells. Here we determined PKC isozyme(s) activated by CSE and their activation mechanism. In C6 glioma cells, mRNAs for PKC alpha, PKC delta, PKC epsilon, and PKC iota were expressed. CSE triggered translocation of PKC alpha and PKC epsilon to plasma membrane. CSE-induced cell damage and PKC translocation were inhibited by chelating intracellular Ca2+ but not extracellular Ca2+. These results suggest that CSE induces cell damage through intracellular Ca2+-dependent activation of PKC alpha and PKC epsilon and subsequent NOX activation.

Different binding property of STIM1 and its novel splice variant STIM1L to Orai1, TRPC3, and TRPC6 channels
Takahiro Horinouchi, Tsunehito Higashi, Tsunaki Higa, Koji Terada, Yosuke Mai, Hiroyuki Aoyagi, Chizuru Hatate, Prabha Nepal, Mika Horiguchi, Takuya Harada, Soichi Miwa
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 428, 2, 252, 258, ACADEMIC PRESS INC ELSEVIER SCIENCE, 2012年11月, ［査読有り］
英語, 研究論文（学術雑誌）, Stromal interaction molecule 1 (STIM1) is the endoplasmic reticulum (ER) Ca2+ sensor to control ER Ca2+ levels. A recent study has shown that STIM1L, a new splice variant of STIM1, is expressed in various tissues of rodent and in human skeletal muscle, and that the interaction of STIM1L with actin filament allows rapid activation of store-operated Ca2+ entry (SOCE) mediated through Orai1 channels. Here, we characterize mRNA expression and function of human STIM1 and STIM1L, and compare their binding property to Orai1 functioning as store-operated Ca2+ channels (SOCCs), and TRPC3 (transient receptor potential canonical 3) and TRPC6 channels functioning as endothelin type A receptor (ETAR)-operated Ca2+ channels (ROCCs). Although mRNA for STIM1 was ubiquitously expressed in human tissues, STIM1L was detected only in skeletal muscle. STIM1L augmented thapsigargin- and endothelin-1 -induced SOCE more strongly than STIM1 in human embryonic kidney 293 cells stably expressing ETAR, whereas, it tends to suppress ETAR-operated Ca2+ entry (ROCE) via TRPC3 and TRPC6 more strongly than STIM1. Coimmunoprecipitation experiments have revealed that when compared with STIM1, STIM1L binds more abundantly to Orai1 and also to TRPC3 and TRPC6. These results suggest that the higher binding capacity of ST1M1L to SOCCs and ROCCs plays an important role in the regulation of Ca2+ signaling such as the augmentation of SOCE via rail and the inhibition of ROCE via TRPC3 and TRPC6. (C) 2012 Elsevier Inc. All rights reserved.

Development of Cysteine-Free Fluorescent Proteins for the Oxidative Environment
Takahisa Suzuki, Seisuke Arai, Mayumi Takeuchi, Chiye Sakurai, Hideaki Ebana, Tsunehito Higashi, Hitoshi Hashimoto, Kiyotaka Hatsuzawa, Ikuo Wada
PLOS ONE, 7, 5, e37551, PUBLIC LIBRARY SCIENCE, 2012年05月, ［査読有り］
英語, 研究論文（学術雑誌）, Molecular imaging employing fluorescent proteins has been widely used to highlight specific reactions or processes in various fields of the life sciences. Despite extensive improvements of the fluorescent tag, this technology is still limited in the study of molecular events in the extracellular milieu. This is partly due to the presence of cysteine in the fluorescent proteins. These proteins almost cotranslationally form disulfide bonded oligomers when expressed in the endoplasmic reticulum (ER). Although single molecule photobleaching analysis showed that these oligomers were not fluorescent, the fluorescent monomer form often showed aberrant behavior in folding and motion, particularly when fused to cysteine-containing cargo. Therefore we investigated whether it was possible to eliminate the cysteine without losing the brightness. By site-saturated mutagenesis, we found that the cysteine residues in fluorescent proteins could be replaced with specific alternatives while still retaining their brightness. cf(cysteine-free) SGFP2 showed significantly reduced restriction of free diffusion in the ER and marked improvement of maturation when fused to the prion protein. We further applied this approach to TagRFP family proteins and found a set of mutations that obtains the same level of brightness as the cysteine-containing proteins. The approach used in this study to generate new cysteine-free fluorescent tags should expand the application of molecular imaging to the extracellular milieu and facilitate its usage in medicine and biotechnology.

RBMX: A Regulator for Maintenance and Centromeric Protection of Sister Chromatid Cohesion
Sachihiro Matsunaga, Hideaki Takata, Akihiro Morimoto, Kayoko Hayashihara, Tsunehito Higashi, Kouhei Akatsuchi, Eri Mizusawa, Mariko Yamakawa, Mamoru Ashida, Tomoko M. Matsunaga, Takachika Azuma, Susumu Uchiyama, Kiichi Fukui
CELL REPORTS, 1, 4, 299, 308, CELL PRESS, 2012年04月, ［査読有り］
英語, 研究論文（学術雑誌）, Cohesion is essential for the identification of sister chromatids and for the biorientation of chromosomes until their segregation. Here, we have demonstrated that an RNA-binding motif protein encoded on the X chromosome (RBMX) plays an essential role in chromosome morphogenesis through its association with chromatin, but not with RNA. Depletion of RBMX by RNA interference (RNAi) causes the loss of cohesin from the centromeric regions before anaphase, resulting in premature chromatid separation accompanied by delocalization of the shugoshin complex and outer kinetochore proteins. Cohesion defects caused by RBMX depletion can be detected as early as the G2 phase. Moreover, RBMX associates with the cohesin subunits, Scc1 and Smc3, and with the cohesion regulator, Wapl. RBMX is required for cohesion only in the presence of Wapl, suggesting that RBMX is an inhibitor of Wapl. We propose that RBMX is a cohesion regulator that maintains the proper cohesion of sister chromatids.

Negative effects of GM-CSF signaling in a murine model of t(8;21)-induced leukemia
Shinobu Matsuura, Ming Yan, Miao-Chia Lo, Eun-Young Ahn, Stephanie Weng, David Dangoor, Mahan Matin, Tsunehito Higashi, Gen-Sheng Feng, Dong-Er Zhang
BLOOD, 119, 13, 3155, 3163, AMER SOC HEMATOLOGY, 2012年03月, ［査読有り］
英語, 研究論文（学術雑誌）, The t(8;21)(q22;q22) is common in adult acute myeloid leukemia (AML). The RUNX1-ETO fusion protein that is expressed by this translocation is poorly leukemogenic and requires additional mutations for transformation. Loss of sex chromosome (LOS) is frequently observed in t(8;21) AML. In the present study, to evaluate whether LOS cooperates with t(8;21) in leukemogenesis, we first used a retroviral transduction/transplantation model to express RUNX1-ETO in hematopoietic cells from XO mice. The low frequency of leukemia in these mice suggests that the potentially critical gene for suppression of t(8;21) leukemia in humans is not conserved on mouse sex chromosomes. The gene encoding the GM-CSF receptor alpha subunit (CSF2RA) is located on X and Y chromosomes in humans but on chromosome 19 in mice. GM-CSF promotes myeloid cell survival, proliferation, and differentiation. To determine whether GM-CSF signaling affects RUNX1-ETOleukemogenesis, hematopoietic stem/progenitor cells that lack GM-CSF signaling were used to express RUNX1-ETO and transplanted into lethally irradiated mice, and a high penetrance of AML was observed in recipients. Furthermore, GM-CSF reduced the replating ability of RUNX1-ETO-expressing cells. These results suggest a possible tumor-suppressor role of GM-CSF in RUNX1-ETO leukemia. Loss of the CSF2RA gene may be a critical mutation explaining the high incidence of LOS associated with the t(8;21)(q22;q22) translocation. (Blood. 2012;119(13):3155-3163)

In vivo manipulation of fluorescently labeled organelles in living cells by multiphoton excitation
Wataru Watanabe, Sachihiro Matsunaga, Tsunehito Higashi, Kiichi Fukui, Kazuyoshi Itoh
JOURNAL OF BIOMEDICAL OPTICS, 13, 3, 031213(1-8), SPIE-SOC PHOTO-OPTICAL INSTRUMENTATION ENGINEERS, 2008年05月
英語, 研究論文（学術雑誌）, Femtosecond laser pulses in the near-infrared region have potential applications in the imaging and manipulation of intracellular organelles. We report on the manipulation of intracellular organelles by two-photon excitation. The dynamics of green fluorescent protein (GFP)-histone are investigated by two-photon fluorescence recovery after photobleaching (FRAP). Intracellular ablation of fluorescently labeled organelles in living cells is performed by focusing femtosecond laser pulses. We report on the selective marking of individual organelles by using two-photon conversion of a photoconvertible fluorescent protein. (c) 2008 Society of Photo-Optical Instrumentation Engineers.

Histone H2A mobility is regulated by its tails and acetylation of core histone tails
Tsunehito Higashi, Sachihiro Matsunaga, Keisuke Isobe, Akihiro Morimoto, Tomoko Shimada, Shogo Kataoka, Wataru Watanabe, Susumu Uchiyama, Kazuyoshi Itoh, Kiichi Fukui
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 357, 3, 627, 632, ACADEMIC PRESS INC ELSEVIER SCIENCE, 2007年06月, ［査読有り］, ［筆頭著者］
英語, 研究論文（学術雑誌）, Historic tail domains play important roles in cellular processes, such as replication, transcription, and chromosome condensation. Histone H2A has one central and two tail domains, and their functions have mainly been studied from a biochemical perspective. In addition, analyses based on visualization have been employed for functional analysis of some chromatin proteins. In this study, we analyzed histone H2A mobility in vivo by two-photon FRAP, and elucidated that the histone H2A N- and C-terminal tails regulate its mobility. We found that histone H2A mobility was increased following treatment of host cells with a histone deacetylase inhibitor. Our results support a model in which core histone tails directly regulate transcription by interacting with nucleosome DNA via electrostatic interactions. (c) 2007 Published by Elsevier Inc.

Development of a multistage classifier for a monitoring system of cell activity based on imaging of chromosomal dynamics
Arni E. Gambe, Rika Maniwa Ono, Sachihiro Matsunaga, Natsumaro Kutsuna, Takumi Higaki, Tsunehito Higashi, Seiichiro Hasezawa, Susumu Uchiyama, Kiichi Fukui
CYTOMETRY PART A, 71A, 5, 286, 296, WILEY-LISS, 2007年05月, ［査読有り］
英語, 研究論文（学術雑誌）, Background: Cell-based assays utilizing digital image cytometry yield multivariate sets of information measuring the efficacy of medicines/chemicals. The use of a HeLa cell line that expresses a GFP-Histone-H1 fusion protein further enhances the performance of these systems, avoiding the use of dyes that may have detrimental influence on cells. Aside from the mitotic index, the distribution of the cell-cycle phases during mitosis can be used as measures of drug/treatment efficacy. Quantification of these parameters, however, requires skill and is time consuming. The purpose of this research was therefore to create a classifier to be incorporated into a system that can automaiically identify the cell-cycle phases in a given image.
Methods: Features based on the shape and texture of the chromosomal regions in images of live Hela cells were measured and analyzed. linear discriminant functions were calculated for the eight cell-cycle phases: interphase, prophase, prometaphase, metaphase, early anaphase, anaphase, telophase and cytokinesis.
Results: The multistage linear discriminant classifier developed had an average classification efficiency of 87-30%.
Conclusion: We demonstrated the possibility of creating a classifier to discriminate between cell-cycle phases using shape and texture features of chromosomal regions. The classifier can be fused to an algorithm for image segmentation, forming a system to automatically and rapidly measure the aforementioned parameters. The results can then be collated to constitute an assay assessing the effects of a drug or treatment on mammalian cells. (c) 2007 International Society for Analytical Cytology.

Stimulated parametric emission microscopy
Isobe K, Kataoka S, Murase R, Watanabe W, Higashi T, Kawakami S, Matsunaga S, Fukui K, Itoh K
Opt Express, 14, 789, 793, 2006年, ［査読有り］

Intracellular disruption of mitochondria in a living HeLa cell with a 76-MHz femtosecond laser oscillator
T Shimada, W Watanabe, S Matsunaga, T Higashi, H Ishii, K Fukui, K Isobe, K Itoh
OPTICS EXPRESS, 13, 24, 9869, 9880, OPTICAL SOC AMER, 2005年11月, ［査読有り］
英語, 研究論文（学術雑誌）, Femtosecond laser pulses can be used to selectively disrupt and dissect intracellular organelles. We report on disruption of mitochondria in living HeLa cells using a femtosecond laser oscillator with a repetition rate of 76 MHz. We studied the laser parameters used for disruption. The long-term viability of the cells after disruption of a single mitochondrion was confirmed by the observation of cell division, indicating that intracellular disruption of organelles using a femtosecond laser oscillator can be performed without compromising the long-term cell viability. (c) 2005 Optical Society of America

Generation of monoclonal antibodies against chromosomal antigens that have a high sequence similarity between human and mouse
T Higashi, S Miyakawa, S Uchiyama, S Matsunaga, H Takata, S Fujimoto, M Noda, A Terauchi, T Shimizu, M Oda, T Azuma, K Fukui
JOURNAL OF BIOTECHNOLOGY, 120, 3, 262, 272, ELSEVIER SCIENCE BV, 2005年11月, ［査読有り］, ［筆頭著者］
英語, 研究論文（学術雑誌）, We raised monoclonal antibodies by immunizing mice with total chromosome proteins extracted from isolated human metaphase chromosomes. The indirect immunofluorescence, screening of hybridoma cell lines provided 15 monoclonal antibodies against the chromosomal antigens. The antigen proteins of the mAbs were identified by immunoblotting as core histones or by immunoprecipitation followed by a peptide mass fingerprinting method as nuclear mitotic apparatus protein, heterogeneous nuclear ribonucleoprotein A2/B1, ribosomal protein S4, linker histone and beta-actin. During mitosis, localizations of these proteins on chromosomes were clearly observed using the obtained antibodies. These results indicate that the current strategy is effective for obtaining monoclonal antibodies useful for immunoblotting and/or immunofluorescent staining of human proteins, using the antigens with high homology to mouse proteins. (c) 2005 Elsevier B.V. All rights reserved.

Femtosecond laser disruption of mitochondria in living cells.
Watanabe, W, Matsunaga, S, Shimada, T, Higashi, T, Fukui, K, Itoh, K
Medical Laser Application, 20, 3, 185, 191, 2005年10月, ［査読有り］
英語

Proteome analysis of human metaphase chromosomes
S Uchiyama, S Kobayashi, H Takata, T Ishihara, N Hori, T Higashi, K Hayashihara, T Sone, D Higo, T Nirasawa, T Takao, S Matsunaga, K Fukui
JOURNAL OF BIOLOGICAL CHEMISTRY, 280, 17, 16994, 17004, AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, 2005年04月, ［査読有り］
英語, 研究論文（学術雑誌）, DNA is packaged as chromatin in the interphase nucleus. During mitosis, chromatin fibers are highly condensed to form metaphase chromosomes, which ensure equal segregation of replicated chromosomal DNA into the daughter cells. Despite > 1 century of research on metaphase chromosomes, information regarding the higher order structure of metaphase chromosomes is limited, and it is still not clear which proteins are involved in further folding of the chromatin fiber into metaphase chromosomes. To obtain a global view of the chromosomal proteins, we performed proteome analyses on three types of isolated human metaphase chromosomes. We first show the results from comparative proteome analyses of two types of isolated human metaphase chromosomes that have been frequently used in biochemical and morphological analyses. 209 proteins were quantitatively identified and classified into six groups on the basis of their known interphase localization. Furthermore, a list of 107 proteins was obtained from the proteome analyses of highly purified metaphase chromosomes, the majority of which are essential for chromosome structure and function. Based on the information obtained on these proteins and on their localizations during mitosis as assessed by immunostaining, we present a four-layer model of metaphase chromosomes. According to this model, the chromosomal proteins have been newly classified into each of four groups: chromosome coating proteins, chromosome peripheral proteins, chromosome structural proteins, and chromosome fibrous proteins. This analysis represents the first compositional view of human metaphase chromosomes and provides a protein framework for future research on this topic.

Multi-spectral two-photon excited fluorescence microscopy using supercontinuum light source
K Isobe, W Watanabe, S Matsunaga, T Higashi, K Fukui, K Itoh
JAPANESE JOURNAL OF APPLIED PHYSICS PART 2-LETTERS & EXPRESS LETTERS, 44, 1-7, L167, L169, INST PURE APPLIED PHYSICS, 2005年, ［査読有り］
英語, 研究論文（学術雑誌）, We report on a novel technique of multi-spectral two-photon-excited fluorescence microscopy by use of broadband supercontinuum light. A supercontinuum in the near-infrared region is generated in a 4.5-min-long photonic crystal fiber using a Ti:sapphire femtosecond oscillator. The supercontinuum is used as a multi-spectral excitation light source in a two-photon excited fluorescence microscope. We demonstrate that three-color fluorescence images of organelles in a cell can be simultaneously acquired.

Stimulated parametric fluorescence microspectroscopy
K. Isobe, R. Murase, S. Kataoka, W. Watanabe, S. Kawakami, S. Matsunaga, T. Higashi, K. Fukui, K. Itoh
Conference Proceedings - Lasers and Electro-Optics Society Annual Meeting-LEOS, 2005, 549, 550, 2005年, ［査読有り］
英語, 研究論文（国際会議プロシーディングス）, We propose a novel technique of four-wave mixing (FWM) microspectroscopy enhanced by a two-photon electronic resonance of pump pulses along with the stimulated emission induced by a dump pulse. © 2005 IEEE.

Cell culture in a closed nano-space
T Doi, S Matsunaga, E Maeno, K Tsuchiya, T Higashi, S Misawa, S Uchiyama, T Ooi, M Nakao, K Fukui
JOURNAL OF BIOSCIENCE AND BIOENGINEERING, 98, 4, 304, 305, SOC BIOSCIENCE BIOENGINEERING JAPAN, 2004年10月, ［査読有り］
英語, 研究論文（学術雑誌）, The minimum size of a closed nano-space in which cells can survive was determined using 4-nl nanowells. One or two cells could divide in the nanowell. Our results suggest that the cell division activity in the nano-space is determined by the conflict between intercellular effects and consumption of substrates.

Femtosecond laser disruption of subcellular organelles in a living cell
W Watanabe, N Arakawa, S Matsunaga, T Higashi, K Fukui, K Isobe, K Itoh
OPTICS EXPRESS, 12, 18, 4203, 4213, OPTICAL SOC AMER, 2004年09月, ［査読有り］
英語, 研究論文（学術雑誌）, Subcellular organelles in living cells were inactivated by tightly focusing femtosecond laser pulses inside the cells. Photodisruption of a mitochondrion in living cells was experimentally confirmed by stacking three-dimensional confocal images and by restaining of organelles. The viability of the cells after femtosecond laser irradiation was ascertained by impermeability of propidium iodide as well as by the presence of cytoplasmic streaming. (C) 2004 Optical Society of America.

An Arabidopsis thaliana gene on the yeast artificial chromosome can be transcribed in tobacco cells
Haibo Liu, Akira Kawabe, Sachihiro Matsunaga, Hee Kim Yeon, Tsunehito Higashi, Susumu Uchiyama, Satoshi Harashima, Akio Kobayashi, Kiichi Fukui
Cytologia, 69, 2, 235, 240, 2004年06月, ［査読有り］
英語, 研究論文（学術雑誌）, A part of chromosome 5 from Arabidopsis thaliana, which has 36 genes, on yeast artificial chromosome (YAC) was transferred into tobacco BY-2 cells using a bio-active beads method. The YAC was constructed using the chromosome-splitting technique. The GFP gene located on the YAC showed repeated transient expression after transformation. Molecular analyses confirmed that 1 (At5g57980) of the 5 genes checked had transcribed in the tobacco BY-2 cells. This demonstrates that the promoter of this gene could work in both A. thaliana and tobacco BY-2 cells.

A novel transfection method for mammalian cells using calcium alginate microbeads
T Higashi, E Nagamori, T Sone, S Matsunaga, K Fukui
JOURNAL OF BIOSCIENCE AND BIOENGINEERING, 97, 3, 191, 195, SOC BIOSCIENCE BIOENGINEERING JAPAN, 2004年03月, ［査読有り］, ［筆頭著者］
英語, 研究論文（学術雑誌）, The direct transfer of genetic materials into mammalian cells is an indispensable technique. We have developed calcium alginate (CA) microbeads which can deliver plasmid DNAs and yeast artificial chromosomes into plant and yeast cells. In this paper, we demonstrate the effective transfection of mammalian cells by CA microbeads immobilizing plasmid DNAs. The transfection was performed using the pEGFP-C1 plasmid containing the cytomegalovirus (CMV) promoter and enhanced green fluorescent protein (EGFP) gene. The transient expression of EGFP was observed 24 h after transfection. The expression efficiency was maximum when the concentration of sodium alginate was 1% and the amount of plasmid DNA was increased to 100 mug. The expression efficiency of our method using CA microbeads is 2-10 times higher than that of the polyethylene glycol (PEG) method. Our results suggest that the CA microbead mediated transfection of mammalian cells effectively delivers genetic materials into mammalian suspension cells.

CLEIAで検出されない抗デスモグレイン3抗体は抗原との結合速度が遅い　　　　　　　　　　　　　　　
眞井 洋輔, 氏家 英之, 東 恒仁, 岩田 浩明, 清水 宏, 日本皮膚科学会雑誌, 128, 4, 607, 607, 2018年04月
(公社)日本皮膚科学会, 日本語

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05月
(一社)日本細胞生物学会, 日本語

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, 2017年

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年

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
AMER SOC CELL BIOLOGY, 英語, 研究発表ペーパー・要旨（国際会議）

エンドセリン受容体シグナルソームの形成を介した新たな2型糖尿病発症メカニズムの解明
堀之内 孝広, 寺田 晃士, 東 恒仁, 医科学応用研究財団研究報告, 32, 320, 324, 2013年
鈴木謙三記念医科学応用研究財団, 日本語

In vivo manipulation of fluorescently labeled organelles in living cells by multiphoton excitation
Wataru Watanabe, Sachihiro Matsunaga, Tsunehito Higashi, Kiichi Fukui, Kazuyoshi Itoh, JOURNAL OF BIOMEDICAL OPTICS, 13, 3, 031213(1-8), 2008年05月
Femtosecond laser pulses in the near-infrared region have potential applications in the imaging and manipulation of intracellular organelles. We report on the manipulation of intracellular organelles by two-photon excitation. The dynamics of green fluorescent protein (GFP)-histone are investigated by two-photon fluorescence recovery after photobleaching (FRAP). Intracellular ablation of fluorescently labeled organelles in living cells is performed by focusing femtosecond laser pulses. We report on the selective marking of individual organelles by using two-photon conversion of a photoconvertible fluorescent protein. (c) 2008 Society of Photo-Optical Instrumentation Engineers., SPIE-SOC PHOTO-OPTICAL INSTRUMENTATION ENGINEERS, 英語

Analyses of human chromosomal proteins using monoclonal antibodies
Tsunehito Higashi, Shuichi Miyakawa, Hideaki Takata, Akiko Terauchi, Takeyuki Shimizu, Masayuki Oda, Susumu Uchiyama, Sachihiro Matsunaa, Takachika Azuma, Kiichi Fukui, CELL STRUCTURE AND FUNCTION, 29, 72, 72, 2004年05月
JAPAN SOC CELL BIOLOGY, 英語, 研究発表ペーパー・要旨（国際会議）

バイオアクティブビーズを用いた動物細胞への遺伝子導入法　　　　　　　　　　　　　　　
東恒仁, 松永幸大, 福井希一, 生体の科学, 55, 2月, 171, 177, 2004年
日本語

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, 2022年
2022年 - 2022年

不飽和カルボニル化合物の毒性メカニズムと生理作用の解明　　　　　　　　　　　　　　　
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, 2021年
2021年 - 2021年

エンドセリン-1によるERKの活性化におけるAnnexin A2の役割　　　　　　　　　　　　　　　
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), 2021年
2021年 - 2021年

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), 2020年
2020年 - 2020年

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, 2019年
2019年 - 2019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), 2019年
2019年 - 2019年

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, 2018年
2018年 - 2018年

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), 2018年
2018年 - 2018年

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), 2018年
2018年 - 2018年

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
第40回日本分子生物学会, 2017年12月, 日本語, ポスター発表
［国内会議］

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, 2017年
2017年 - 2017年

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, 2017年
2017年 - 2017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), 2017年
2017年 - 2017年

Unsaturated carbonyl compounds in the gas phase extract of cigarette smoke induce cell death through intracellular Ca2+-dependent PKC activation　　　　　　　　　　　　　　　
Tsunehito Higashi, Yosuke Mai, Yuichi Mazaki, Takahiro Horinouchi, Soichi Miwa
ASCB 2016 Annual Meeting, 2016年12月04日, 英語, ポスター発表
［国際会議］

タバコ煙ガス相による細胞傷害の抑制因子の探索と抑制メカニズムの解明　　　　　　　　　　　　　　　
東恒仁, 眞井洋輔, 堀之内孝広, 真崎雄一, 三輪聡一
第89回日本薬理学会年会, 2016年03月09日, 日本語, 口頭発表（一般）
［国内会議］

ニコチン及びタール除去タバコ煙抽出物の細胞傷害作用に関与するPKCアイソザイムの同定と活性化機構の解明　　　　　　　　　　　　　　　
東 恒仁, 眞井洋輔, 寺田晃士, 旗手千鶴, 堀之内孝広, 三輪聡一
第63回日本薬理学会北部会, 2012年09月14日, 日本語, 口頭発表（一般）
［国内会議］

DCP1のP-bodyへの局在にはそのC末端領域が必要である　　　　　　　　　　　　　　　
第22回日本動物細胞工学会大会, 2009年, ポスター発表

Role of Dcp1 in the formation of P-bodies　　　　　　　　　　　　　　　
Biophysical Society 53rd Annual Meeting, 2009年, ポスター発表

P-body形成におけるDCP1の役割　　　　　　　　　　　　　　　
第31回日本分子生物学会年会, 2008年, ポスター発表

二光子レーザー顕微法を用いた細胞小器官の操作　　　　　　　　　　　　　　　
第20回動物細胞工学シンポジウム, 2008年

3C11-2 テールドメインによるヒストンH2Aの細胞内動態制御(酵素学・酵素工学・タンパク質工学,その他,一般講演)
東 恒仁, 森本 晃弘, 磯部 圭祐, 渡辺 歴, 内山 進, 松永 幸大, 伊東 一良, 福井 希一
日本生物工学会大会講演要旨集, 2007年, 日本生物工学会, 日本語
2007年 - 2007年

テールドメインによるヒストンH2Aの細胞内動態制御　　　　　　　　　　　　　　　
第59回日本生物工学会大会, 2007年

小胞体内におけるカーゴ蛋白質一分子ダイナミクスの解析　　　　　　　　　　　　　　　
第30回日本分子生物学会年会, 2007年

2H11-5 GFPを用いた染色体タンパク質の局在およびFRAP解析(遺伝子工学,核酸工学,一般講演)
森本 晃弘, 東 恒仁, 真庭 理香, 内山 進, 渡辺 歴, 松永 幸大, 伊東 一良, 福井 希一
日本生物工学会大会講演要旨集, 2006年, 日本生物工学会, 日本語
2006年 - 2006年

GFPを用いた染色体タンパク質の局在およびFRAP解析　　　　　　　　　　　　　　　
平成18年度日本生物工学会大会, 2006年

Mobility analyses of linker and core histones by two-photon FRAP　　　　　　　　　　　　　　　
20th IUBMB, 2006年, ポスター発表

1H16-5 フェムト秒レーザー照射によるミトコンドリアの形態変化(動物細胞工学・動物組織培養,一般講演)
石井 光, 東 恒仁, 島田 朋子, 渡辺 歴, 松永 幸大, 伊東 一良, 福井 希一
日本生物工学会大会講演要旨集, 2005年, 日本生物工学会, 日本語
2005年 - 2005年

1H16-4 二光子FRAPを用いたタンパク質の細胞内動態解析(動物細胞工学・動物組織培養,一般講演)
東 恒仁, 磯辺 圭祐, 渡辺 歴, 松永 幸大, 伊東 一良, 福井 希一
日本生物工学会大会講演要旨集, 2005年, 日本生物工学会, 日本語
2005年 - 2005年

モノクローナル抗体を用いた染色体局在タンパク質の同定および解析　　　　　　　　　　　　　　　
宮川秀一, 東恒仁, 藤本聡, 内山進, 松永幸大, 福井希一
日本分子生物学会年会プログラム・講演要旨集, 2004年
2004年 - 2004年

3S2-AM2 レーザー3次元顕微鏡を用いた生体内1分子動態の可視化(レーザーマイクロマニピュレーションを用いた生物機能解明-その現状と展望-,シンポジウム)
福井 希一, 松永 幸大, 東 恒仁, 磯部 圭佑, 渡辺 歴, 伊東 一良
日本生物工学会大会講演要旨集, 2004年, 日本生物工学会, 日本語
2004年 - 2004年

3C11-2 バイオアクティブビーズを用いた動物細胞への遺伝子導入法の開発(遺伝子工学・核酸工学,一般講演)
馬場 明子, 東 恒仁, 内山 進, 松永 幸大, 福井 希一
日本生物工学会大会講演要旨集, 2004年, 日本生物工学会, 日本語
2004年 - 2004年

看護薬理学　　　　　　　　　　　　　　　
天使大学看護栄養学部

薬理学　　　　　　　　　　　　　　　
北海道大学医学部、日本医療大学保健医療学部, 名寄市立大学保健福祉学部

薬理学実習　　　　　　　　　　　　　　　
北海道大学医学部

臨床薬理学　　　　　　　　　　　　　　　
日本医療大学保健医療学部

不飽和カルボニル化合物によるPKCを介した新しいフェロトーシス誘導機序の解明
科学研究費助成事業
2021年04月 - 2024年03月
東 恒仁
不飽和カルボニル化後物は、有機化合物の燃焼によって発生する環境毒性物質である。不飽和カルボニル化合物は、肺でのガス交換を介して生体に取り込まれ、生理機能に影響を及ぼすと考えられている。したがって、生体内において不飽和カルボニル化合物に曝露されやすい器官は、気管や肺、血管、血液系細胞などではないかと考えられる。そこで、令和3年度には、不飽和カルボニル化合物や、不飽和カルボニル化合物を高濃度に含むタバコ煙が、気管上皮細胞に与える影響について検討した。その結果、タバコ煙ガス相やアクロレイン・メチルビニルケトンなどの不飽和カルボニル化合物が、気管上皮培養細胞において細胞死を引き起こすことがわかった。様々な細胞死に対する阻害薬を用いた検討を行ったところ、タバコ煙ガス相・アクロレイン・メチルビニルケトンによって誘導される細胞死はフェロトーシスであることが分かった。更に、プロテインキナーゼC（PKC）阻害薬によってこのフェロトーシス誘導が抑制されたことから、PKCの関与が強く疑われた。PKCは、10種類のアイソフォームを持つことが知られていることから、どのアイソフォームが本プロセスに関与するかを明らかにするため、アイソフォーム特異的の高いPKC阻害薬を用いた薬理学的解析を実施した。その結果、気管上皮細胞においてフェロトーシスの誘導に関与するPKCは、novel PKCもしくはatypical PKCに分類されるアイソフォームである可能性が高いことが判明した。
日本学術振興会, 基盤研究(C), 北海道大学, 21K12253

不飽和カルボニル化合物による細胞傷害と病態発症におけるプロテインキナーゼCの役割
科学研究費助成事業
2018年04月 - 2021年03月
東 恒仁
人の生活環境には様々な化学物質が存在し、人の健康に影響を与えている。不飽和カルボニル化合物は、主に有機化合物の燃焼によって発生する化学物質であり、強い毒性を持つことが知られているが、その毒性メカニズムは分かっていなかった。本研究では、不飽和カルボニル化合物が、細胞内の特定のシグナル伝達経路を活性化することで細胞死を引き起こしてることを明らかにした。更に、アミノ酸であるシステインやシステインの誘導体が、不飽和カルボニル化合物と直接反応してその細胞毒性を抑制することを示した。
日本学術振興会, 基盤研究(C), 北海道大学, 18K11654

タバコ煙中の細胞傷害因子による動脈硬化発症機構の解明と細胞傷害因子除去法の確立
科学研究費助成事業 若手研究(B)
2014年04月 - 2017年03月
東 恒仁, 眞井 洋輔
喫煙は動脈硬化症などの心血管系疾患の危険因子である。本研究では、タバコ煙ガス相に含まれる不飽和カルボニル化合物の病態生理学的意義の解明とその影響の抑制方法の開発を目的とした。タバコ煙ガス相に含まれる不飽和カルボニル化合物は、細胞内カルシウム依存的にプロテインキナーゼCを活性化し、細胞傷害を引き起こすことを見出した。また、還元型グルタチオンやビタミンEなどの抗酸化物質が効果的に不飽和カルボニル化合物による細胞傷害を抑制できることが判明した。
日本学術振興会, 若手研究(B), 北海道大学, 26860166

細胞の品質管理機構に着目したバイオ医薬品の生産性向上に関する研究
科学研究費助成事業
2008年 - 2009年
東 恒仁, 和田 郁夫
小胞体内腔のタンパク質の動態を解析するために、全反射顕微鏡を用いた一分子観察の系を確立した。一分子解析は得られるデータが膨大なものとなるが、それらを解析するために画像解析の手法を導入した。またこれまで当研究室で行われてきた解像度を時空間方向に一桁程度高めることで、細胞質で最も速い単純拡散する分子の解析を可能とした。そして確立した手法を用いて、分泌タンパク質の挙動に影響を与える因子の一つとして温度ストレスを見出した。
日本学術振興会, 若手研究(B), 福島県立医科大学, 20760541

成熟過程における分泌系カーゴタンパク質のゲーティング・パーミッション機構の研究
科学研究費助成事業
2007年 - 2008年
和田 郁夫, 初沢 清隆, 橋本 仁志, 中西 英樹, 東 恒仁
なぜほ乳類の細胞ではきわめて高い効率で高品位な分泌が可能となるかを知るために、輸送されるカーゴタンパク質の成熟過程での動態を一分子レベルで計測するシステムを作成した。解析の結果、分子の成熟化を行う小胞体内腔ではカーゴの糖鎖をグリップとして細胞質のアクチン骨格を利用した強い拡散抑制を行うことなどを見いだした。この拡散抑制は高温ストレス下で顕著となり低温ではほとんど観測されないことから、分子成熟化の基盤をなすと考えられる。
日本学術振興会, 基盤研究(B), 公立大学法人福島県立医科大学, 19370044

ヒト人工染色体の細胞への導入方法の確立およびその応用に関する生物工学的研究
科学研究費助成事業
2005年 - 2006年
東 恒仁
アルギン酸カルシウムマイクロビーズを介したヒト人工染色体の細胞への導入方法を確立するにあたり、ヒト人工染色体のvitroでの安定性を高めることが不可欠であることが判明した。そのため、染色体の形態安定に寄与するタンパク質の探索を試みた。前年度までにヒト染色体を構成するタンパク質のカタログ化に成功していることから、これらのタンパク質のノックアウトを行ない、その表現型を観察することで染色体の安定化に寄与する因子の特定を試みた。ノックアウトの容易さ、および実験の速やかさから、分裂酵母Schizosaccharomyces pombeを宿主として用いることとし、ヒト染色体タンパク質の分裂酵母ホモログ約80種類の細胞内局在を調べた後、染色体に局在が見られたタンパク質を順次ノックアウトした。その結果ヒト・分裂酵母でFACT complexを形成するSSRP1の分裂酵母ホモログをノックアウトした場合に細胞分裂に重篤な障害が発生することが判明した。SSRP1と共にFACTを形成するSPT16の分裂酵母ホモログのノックアウトも試みたがノックアウトラインは得られなかった。
SSRP1ノックアウトラインでは細胞分裂速度の低下、クロマチン構造の変化が見られた。
SSRP1と相互作用するタンパク質を同定するために、two-hybrid、およびtandem affinity purification tagを用いたタンパク質精製を行なった。その結果、分裂酵母SSRP1と分裂酵母SPT16との相互作用が確認されたことから、本研究までFACTが同定されていなかった分裂酵母において、初めてFACTの存在を示した。また分裂酵母FACTと相互作用するタンパク質として、apm-1、elongation factor1-α、RNA polymerase subunitなどが同定された。
本研究により分裂酵母においてFACTがクロマチン構造の安定性に寄与していることが示唆された。FACTは保存性が非常に高いタンパク質の複合体であり、ヒトなどの高等真核生物においてもクロマチン・染色体構造の安定性や染色体分配に寄与している可能性が高いと推定されることから、ヒト人工染色体をvitroで扱うにあたり操作性を向上させることができる因子としての役割が期待される。
日本学術振興会, 特別研究員奨励費, 05J09774