Researcher Profile and Settings

Master

Affiliation (Master)

• Faculty of Medicine　Physiological Science　Physiology

Affiliation (Master)

• Faculty of Medicine　Physiological Science　Physiology

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Profile and Settings

Degree

• M.D., Ph.D.（Hokkaido University）

Profile and Settings

• Name (Japanese)

  Ohba

• Name (Kana)

  Yusuke

• Name

  200901014201313812

Alternate Names

http://cp.med.hokudai.ac.jp
http://cp.med.hokudai.ac.jp/en/

Achievement

Research Interests

• 細胞膜動態   Atomic force microscopy   FRET   GFP   bioimaging   Ras   細胞内情報伝達   イメージング   環境適応   生細胞   BiFC   多分子複合体   Signal transduction   Gタンパク質   分子動態   エンドサイトーシス   

Research Areas

• Life sciences / Physiology
• Life sciences / Cell biology
• Life sciences / Experimental pathology

Research Experience

• 2006/04 - 2012/10 Hokkaido University Graduate School of Medicine Associate Professor
• 2004/03 - 2006/03 Graduated School of Medicine, the University of Tokyo Assistant Professor
• 2001/12 - 2005/03 JST PRESTO Researcher
• 2001/01 - 2004/03 Research Institute for Infectious Diseases, Osaka University Research Associate
• 1998/04 - 2000/12 International Medical Center of Japan Research Institute

Education

• 1996/04 - 2000/03  Hokkaido University  Graduated School of Medicine
•        - 1996/03  Hokkaido University  School of Medicine

Awards

• 2013/06 German Research and Innovation Forum Tokyo/German Chamber of Commerce and Industry in Japan German Innovation Award Gottfried Wagner Prize 2013
   FRET-Based Biosensors for Accurate Prediction of Responses to Molecular Target Drugs in Chronic Myeloid Leukemia 

  受賞者: OHBA Yusuke

Published Papers

• Intravenous Administration of Mesenchymal Stem Cell-Derived Exosome Alleviates Spinal Cord Injury by Regulating Neutrophil Extracellular Trap Formation through Exosomal miR-125a-3p.

  Yutaka Morishima, Masahito Kawabori, Kazuyoshi Yamazaki, Soichiro Takamiya, Sho Yamaguchi, Yo Nakahara, Hajime Senjo, Daigo Hashimoto, Sakiko Masuda, Yoichiro Fujioka, Yusuke Ohba, Yuki Mizuno, Yuji Kuge, Miki Fujimura

  International journal of molecular sciences 25 (4) 2024/02/18 

  Spinal cord injury (SCI) leads to devastating sequelae, demanding effective treatments. Recent advancements have unveiled the role of neutrophil extracellular traps (NETs) produced by infiltrated neutrophils in exacerbating secondary inflammation after SCI, making it a potential target for treatment intervention. Previous research has established that intravenous administration of stem cell-derived exosomes can mitigate injuries. While stem cell-derived exosomes have demonstrated the ability to modulate microglial reactions and enhance blood-brain barrier integrity, their impact on neutrophil deactivation, especially in the context of NETs, remains poorly understood. This study aims to investigate the effects of intravenous administration of MSC-derived exosomes, with a specific focus on NET formation, and to elucidate the associated molecular mechanisms. Exosomes were isolated from the cell supernatants of amnion-derived mesenchymal stem cells using the ultracentrifugation method. Spinal cord injuries were induced in Sprague-Dawley rats (9 weeks old) using a clip injury model, and 100 μg of exosomes in 1 mL of PBS or PBS alone were intravenously administered 24 h post-injury. Motor function was assessed serially for up to 28 days following the injury. On Day 3 and Day 28, spinal cord specimens were analyzed to evaluate the extent of injury and the formation of NETs. Flow cytometry was employed to examine the formation of circulating neutrophil NETs. Exogenous miRNA was electroporated into neutrophil to evaluate the effect of inflammatory NET formation. Finally, the biodistribution of exosomes was assessed using 64Cu-labeled exosomes in animal positron emission tomography (PET). Rats treated with exosomes exhibited a substantial improvement in motor function recovery and a reduction in injury size. Notably, there was a significant decrease in neutrophil infiltration and NET formation within the spinal cord, as well as a reduction in neutrophils forming NETs in the circulation. In vitro investigations indicated that exosomes accumulated in the vicinity of the nuclei of activated neutrophils, and neutrophils electroporated with the miR-125a-3p mimic exhibited a significantly diminished NET formation, while miR-125a-3p inhibitor reversed the effect. PET studies revealed that, although the majority of the transplanted exosomes were sequestered in the liver and spleen, a notably high quantity of exosomes was detected in the damaged spinal cord when compared to normal rats. MSC-derived exosomes play a pivotal role in alleviating spinal cord injury, in part through the deactivation of NET formation via miR-125a-3p.
• 血管内皮細胞におけるSARS-CoV-2侵入機構の解析(Analysis of SARS-CoV-2 uptake mechanism in vascular endothelial cells)

  桜井 優弥, 間石 奈湖, 藤岡 容一郎, 大場 雄介, 武田 遼, 佐々木 道仁, 大場 靖子, 樋田 泰浩, 澤 洋文, 樋田 京子

  日本病理学会会誌 113 (1) 369 - 369 0300-9181 2024/02
• Development of Polymer-Lipid Hybrid Nanoparticles for Large-Sized Plasmid DNA Transfection.

  Masatoshi Maeki, Shuya Uno, Kaisei Sugiura, Yusuke Sato, Yoichiro Fujioka, Akihiko Ishida, Yusuke Ohba, Hideyoshi Harashima, Manabu Tokeshi

  ACS applied materials & interfaces 16 (2) 2110 - 2119 2024/01/17 

  RNA and DNA delivery technologies using lipid nanoparticles (LNPs) have advanced significantly, as demonstrated by their successful application in mRNA vaccines. To date, commercially available RNA therapeutics include Onpattro, a 21 bp siRNA, and mRNA vaccines comprising 4300 nucleotides for COVID-19. However, a significant challenge remains in achieving efficient transfection, as the size of the delivered RNA and DNA increases. In contrast to RNA transfection, plasmid DNA (pDNA) transfection requires multiple steps, including cellular uptake, endosomal escape, nuclear translocation, transcription, and translation. The low transfection efficiency of large pDNA is a critical limitation in the development of artificial cells and their cellular functionalization. Here, we introduce polymer-lipid hybrid nanoparticles designed for efficient, large-sized pDNA transfection. We demonstrated that LNPs loaded with positively charged pDNA-polycation core nanoparticles exhibited a 4-fold increase in transfection efficiency for 15 kbp pDNA compared with conventional LNPs, which encapsulate a negatively charged pDNA-polycation core. Based on assessments of the size and internal structure of the polymer-lipid nanoparticles as well as hemolysis and cellular uptake analysis, we propose a strategy to enhance large-sized pDNA transfection using LNPs. This approach holds promise for accelerating the in vivo delivery of large-sized pDNA and advancing the development of artificial cells.
• Strength in numbers: Unleashing the potential of trans-scale scope AMATERAS for massive cell quantification

  Taro Ichimura, Taishi Kakizuka, Yuki Sato, Yoichiro Fujioka, Yusuke Ohba, Kazuki Horikawa, Takeharu Nagai

  Biophysics and Physicobiology 2024
• Safe and efficient oral allergy immunotherapy using one-pot-prepared mannan-coated allergen nanoparticles.

  Shunyi Li, Hiroki Toriumi, Daisuke Takahashi, Tomoko Kamasaki, Yoichiro Fujioka, Satoru Nagatoishi, Jinting Li, Yiwei Liu, Takanatsu Hosokawa, Kouhei Tsumoto, Yusuke Ohba, Yoshiki Katayama, Daisuke Murakami, Koji Hase, Takeshi Mori

  Biomaterials 303 122381 - 122381 2023/12 

  Allergen immunotherapy (AIT) is the only curative treatment for allergic diseases. However, AIT has many disadvantages related to efficiency, safety, long-term duration, and patient compliance. Dendritic cells (DCs) have an important role in antigen-specific tolerance induction; thus, DC-targeting strategies to treat allergies such as glutaraldehyde crosslinked antigen to mannoprotein (MAN) have been established. However, glutaraldehyde crosslinking may reduce the antigen presentation efficiency of DCs. To overcome this, we developed a MAN-coated ovalbumin (OVA) nanoparticle (MDO), which uses intermolecular disulfide bond to crosslink OVA and MAN. MDO effectively targeted DCs resulting in tolerogenic DCs, and promoted higher antigen presentation efficiency by DCs compared with OVA or glutaraldehyde crosslinked nanoparticles. In vitro and in vivo experiments showed that DCs exposed to MDO induced Treg cells. Moreover, MDO had low reactivity with anti-OVA antibodies and did not induce anaphylaxis in allergic mice, demonstrating its high safety profile. In a mouse model of allergic asthma, MDO had significant preventative and therapeutic effects when administered orally or subcutaneously. Therefore, MDO represents a promising new approach for the efficient and safe treatment of allergies.
• Comparative pathogenicity of SARS-CoV-2 Omicron subvariants including BA.1, BA.2, and BA.5.

  Tomokazu Tamura, Daichi Yamasoba, Yoshitaka Oda, Jumpei Ito, Tomoko Kamasaki, Naganori Nao, Rina Hashimoto, Yoichiro Fujioka, Rigel Suzuki, Lei Wang, Hayato Ito, Yukie Kashima, Izumi Kimura, Mai Kishimoto, Masumi Tsuda, Hirofumi Sawa, Kumiko Yoshimatsu, Yuki Yamamoto, Tetsuharu Nagamoto, Jun Kanamune, Yutaka Suzuki, Yusuke Ohba, Isao Yokota, Keita Matsuno, Kazuo Takayama, Shinya Tanaka, Kei Sato, Takasuke Fukuhara

  Communications biology 6 (1) 772 - 772 2023/07/24 

  The unremitting emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants necessitates ongoing control measures. Given its rapid spread, the new Omicron subvariant BA.5 requires urgent characterization. Here, we comprehensively analyzed BA.5 with the other Omicron variants BA.1, BA.2, and ancestral B.1.1. Although in vitro growth kinetics of BA.5 was comparable among the Omicron subvariants, BA.5 was much more fusogenic than BA.1 and BA.2. Airway-on-a-chip analysis showed that, among Omicron subvariants, BA.5 had enhanced ability to disrupt the respiratory epithelial and endothelial barriers. Furthermore, in our hamster model, in vivo pathogenicity of BA.5 was slightly higher than that of the other Omicron variants and less than that of ancestral B.1.1. Notably, BA.5 gains efficient virus spread compared with BA.1 and BA.2, leading to prompt immune responses. Our findings suggest that BA.5 has low pathogenicity compared with the ancestral strain but enhanced virus spread /inflammation compared with earlier Omicron subvariants.
• Interaction between PI3K and the VDAC2 channel tethers Ras-PI3K-positive endosomes to mitochondria and promotes endosome maturation.

  Aya O Satoh, Yoichiro Fujioka, Sayaka Kashiwagi, Aiko Yoshida, Mari Fujioka, Hitoshi Sasajima, Asuka Nanbo, Maho Amano, Yusuke Ohba

  Cell reports 42 (3) 112229 - 112229 2023/03/28 

  Intracellular organelles of mammalian cells communicate with one another during various cellular processes. The functions and molecular mechanisms of such interorganelle association remain largely unclear, however. We here identify voltage-dependent anion channel 2 (VDAC2), a mitochondrial outer membrane protein, as a binding partner of phosphoinositide 3-kinase (PI3K), a regulator of clathrin-independent endocytosis downstream of the small GTPase Ras. VDAC2 tethers endosomes positive for the Ras-PI3K complex to mitochondria in response to cell stimulation with epidermal growth factor and promotes clathrin-independent endocytosis, as well as endosome maturation at membrane association sites. With an optogenetics system to induce mitochondrion-endosome association, we find that, in addition to its structural role in such association, VDAC2 is functionally implicated in the promotion of endosome maturation. The mitochondrion-endosome association thus plays a role in the regulation of clathrin-independent endocytosis and endosome maturation.
• Secretory glycoprotein NS1 plays a crucial role in the particle formation of flaviviruses

  Tomokazu Tamura, Shiho Torii, Kentaro Kajiwara, Itsuki Anzai, Yoichiro Fujioka, Kisho Noda, Shuhei Taguwa, Yuhei Morioka, Rigel Suzuki, Yuzy Fauzyah, Chikako Ono, Yusuke Ohba, Masato Okada, Takasuke Fukuhara, Yoshiharu Matsuura

  PLOS Pathogens 18 (6) e1010593 - e1010593 2022/06/03 

  Flaviviruses, which are globally distributed and cause a spectrum of potentially severe illnesses, pose a major threat to public health. Although Flaviviridae viruses, including flaviviruses, possess similar genome structures, only the flaviviruses encode the non-structural protein NS1, which resides in the endoplasmic reticulum (ER) and is secreted from cells after oligomerization. The ER-resident NS1 is known to be involved in viral genome replication, but the essential roles of secretory NS1 in the virus life cycle are not fully understood. Here we characterized the roles of secretory NS1 in the particle formation of flaviviruses. We first identified an amino acid residue essential for the NS1 secretion but not for viral genome replication by using protein-protein interaction network analyses and mutagenesis scanning. By using the recombinant flaviviruses carrying the identified NS1 mutation, we clarified that the mutant flaviviruses employed viral genome replication. We then constructed a recombinant NS1 with the identified mutation and demonstrated by physicochemical assays that the mutant NS1 was unable to form a proper oligomer or associate with liposomes. Finally, we showed that the functions of NS1 that were lost by the identified mutation could be compensated for by the in trans-expression of E^rns of pestiviruses and host exchangeable apolipoproteins, which participate in the infectious particle formation of pestiviruses and hepaciviruses in the family Flaviviridae, respectively. Collectively, our study suggests that secretory NS1 plays a role in the particle formation of flaviviruses through its interaction with the lipid membrane.
• Stimulation of the mitochondrial calcium uniporter mitigates chronic heart failure-associated ventricular arrhythmia in mice.

  Hikaru Hagiwara, Masaya Watanabe, Yoichiro Fujioka, Takahide Kadosaka, Takuya Koizumi, Taro Koya, Motoki Nakao, Rui Kamada, Taro Temma, Kazufumi Okada, Jose Antonio Moreno, Ohyun Kwon, Hisakata Sabe, Yusuke Ohba, Toshihisa Anzai

  Heart rhythm 19 (10) 1725 - 1735 2022/05/31 

  BACKGROUND: An aberrant increase in the diastolic calcium concentration ([Ca2+]i) level is a hallmark of heart failure (HF) and the cause of delayed afterdepolarization and ventricular arrhythmia (VA). Although mitochondria play a role in regulating [Ca2+]i, whether they can compensate for the [Ca2+]i abnormality in ventricular myocytes is unknown. OBJECTIVE: We investigated whether enhanced Ca2+ uptake of mitochondria may compensate for an abnormal increase in the [Ca2+]i of ventricular myocytes in HF to effectively mitigate VA. METHODS: We used a HF mouse model, in which myocardial infarction was induced by permanent left anterior descending coronary artery ligation. The mitochondrial Ca2+ uniporter was stimulated by kaempferol. Ca2+ dynamics and membrane potential were measured using an epifluorescence microscope, a confocal microscope, and the perforated patch-clamp technique. VA was induced in the Langendorff-perfused hearts, and the hemodynamic parameters were measured using a microtip transducer catheter. RESULTS: Protein expression of the mitochondrial Ca2+ uniporter, as assessed by its subunit expression, did not change between HF and sham mice. Treatment of cardiomyocytes with kaempferol, isolated from HF mice at 28 days after coronary ligation, reduced the appearance of aberrant diastolic [Ca2+]i waves and sparks and spontaneous action potentials. Kaempferol effectively reduced the VA occurring in Langendorff-perfused hearts. Intravenous administration of kaempferol did not markedly affect the left ventricular hemodynamic parameters. CONCLUSION: The effects of kaempferol in HF of mice implied that mitochondria may have the potential to compensate for abnormal [Ca2+]i. Mechanisms involved in mitochondrial Ca2+ uptake may provide novel targets to treat HF-associated VA.
• A method for the generation of pseudovirus particles bearing SARS coronavirus spike protein in high yields.

  Yoichiro Fujioka, Sayaka Kashiwagi, Aiko Yoshida, Aya O Satoh, Mari Fujioka, Maho Amano, Yohei Yamauchi, Yusuke Ohba

  Cell structure and function 47 (1) 43 - 53 2022/04/28 

  The ongoing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has threatened human health and the global economy. Development of additional vaccines and therapeutics is urgently required, but such development with live virus must be conducted with biosafety level 3 confinement. Pseudotyped viruses have been widely adopted for studies of virus entry and pharmaceutical development to overcome this restriction. Here we describe a modified protocol to generate vesicular stomatitis virus (VSV) pseudotyped with SARS-CoV or SARS-CoV-2 spike protein in high yield. We found that a large proportion of pseudovirions produced with the conventional transient expression system lacked coronavirus spike protein at their surface as a result of inhibition of parental VSV infection by overexpression of this protein. Establishment of stable cell lines with an optimal expression level of coronavirus spike protein allowed the efficient production of progeny pseudoviruses decorated with spike protein. This improved VSV pseudovirus production method should facilitate studies of coronavirus entry and development of antiviral agents.Keywords: severe acute respiratory syndrome coronavirus (SARS-CoV), SARS-CoV-2, pseudovirus, vesicular stomatitis virus (VSV), spike protein.
• Direct visualization of glucagon-like peptide-1 secretion by fluorescent fusion proteins.

  Atsushi Tsuzuki, Yoichiro Fujioka, Aiko Yoshida, Sayaka Kashiwagi, Maho Amano, Tohru Hira, Akinobu Nakamura, Hideaki Miyoshi, Tatsuya Atsumi, Yusuke Ohba

  Journal of diabetes investigation 13 (7) 1134 - 1139 2022/04/04 

  Live-cell imaging with fluorescent proteins (FPs) is a powerful tool for investigating the exocytosis processes of hormones. However, the secretion process of glucagon-like peptide-1 (GLP-1) has not been visualized by FPs, which might be because tagging FPs inhibits GLP-1 synthesis through the post-translational processing from proglucagon. Here, we have developed FP-tagged GLP-1 by inserting FPs into the middle of GLP-1 and adding the proglucagon signal peptide. Confocal imaging confirmed that GLP-1 fused to FPs with high folding efficiency showed granular structure, in which secretory vesicle markers colocalized. The fluorescence intensity of FP in the culture supernatant from cells treated with KCl or forskolin was significantly increased compared with those from untreated cells. Furthermore, FP-tagged GLP-1 enables direct visualization of stimulation-dependent exocytosis of GLP-1 at a single granule resolution with total internal reflection fluorescence microscopy. FP-tagged GLP-1 might facilitate the screening of GLP-1 secretagogues and the discovery of new antidiabetic drugs.
• Imaging technology that enables simultaneous visualization of weak interaction interfaces and cell responses

  Aiko Yoshida, Yoichiro Fujioka, Maho Amano, Yusuke Ohba

  Drug Delivery System 37 (2) 102 - 111 0913-5006 2022/03/25
• Schlafen family member 11 indicates favorable prognosis of patients with head and neck cancer following platinum-based chemoradiotherapy.

  Seijiro Hamada, Satoshi Kano, Junko Murai, Takayoshi Suzuki, Nayuta Tsushima, Takatsugu Mizumachi, Masanobu Suzuki, Tsuyoshi Takashima, Daiki Taniyama, Naoya Sakamoto, Yoichiro Fujioka, Yusuke Ohba, Akihiro Homma

  Frontiers in oncology 12 978875 - 978875 2022 

  Recently, Schlafen family member 11 (SLFN11) has been reported to increase the sensitivity of cancer cells to DNA-damaging agents, including platinum derivatives; thus, SLFN11 may be a predictive biomarker for platinum-based chemoradiotherapy (CRT). In this study, we examined whether SLFN11 expression was associated with the therapeutic outcome of platinum-based CRT in head and neck squamous cell carcinoma (HNSCC). We performed immunohistochemical analyses for SLFN11 expression in 161 HNSCC tissues from patients who had been administered cisplatin-based CRT and examined the correlation between SLFN11 expression and progression-free survival (PFS). Additionally, SLFN11 expression was examined in 10 paired samples obtained before and after CRT in patients with local failure. Furthermore, in vitro experiments were performed using several HNSCC cell lines and isogenic SLFN11-knockout cells to assess the association between SLFN11 expression and drug sensitivity. PFS was found to be significantly better in the SLFN11-positive group than in the SLFN11-negative group among the 161 patients (5-year PFS: 78.8% vs. 52.8%, respectively, p < 0.001). Similar results were observed for the PFS at each primary site. The percentage of SLFN11 positivity was lower in tumor samples from patients with local failure after CRT than that in the corresponding primary tumors before CRT in 8 of 10 cases. Results of the in vitro assay demonstrated that SLFN11-knockout cells exhibited reduced sensitivity to DNA-damaging agents but not to the non-DNA-damaging agent docetaxel. Our findings suggest that SLFN11 may serve as a potential biomarker for predicting the response of HNSCC patients to platinum-based CRT.
• A phospho-switch controls RNF43-mediated degradation of Wnt receptors to suppress tumorigenesis

  Tadasuke Tsukiyama, Juqi Zou, Jihoon Kim, Shohei Ogamino, Yuki Shino, Takamasa Masuda, Alessandra Merenda, Masaki Matsumoto, Yoichiro Fujioka, Tomonori Hirose, Sayuri Terai, Hidehisa Takahashi, Tohru Ishitani, Keiichi I. Nakayama, Yusuke Ohba, Bon-Kyoung Koo, Shigetsugu Hatakeyama

  Nature Communications 11 (1) 2020/12 

  Frequent mutation of the tumour suppressor RNF43 is observed in many cancers, particularly colon malignancies. RNF43, an E3 ubiquitin ligase, negatively regulates Wnt signalling by inducing degradation of the Wnt receptor Frizzled. In this study, we discover that RNF43 activity requires phosphorylation at a triplet of conserved serines. This phospho-regulation of RNF43 is required for zebrafish development and growth of mouse intestinal organoids. Cancer-associated mutations that abrogate RNF43 phosphorylation cooperate with active Ras to promote tumorigenesis by abolishing the inhibitory function of RNF43 in Wnt signalling while maintaining its inhibitory function in p53 signalling. Our data suggest that RNF43 mutations cooperate with KRAS mutations to promote multi-step tumorigenesis via the Wnt-Ras-p53 axis in human colon cancers. Lastly, phosphomimetic substitutions of the serine trio restored the tumour suppressive activity of extracellular oncogenic mutants. Therefore, harnessing phospho-regulation of RNF43 might be a potential therapeutic strategy for tumours with RNF43 mutations.
• Calcium Wave Promotes Cell Extrusion.

  Yasuto Takeuchi, Rika Narumi, Ryutaro Akiyama, Elisa Vitiello, Takanobu Shirai, Nobuyuki Tanimura, Keisuke Kuromiya, Susumu Ishikawa, Mihoko Kajita, Masazumi Tada, Yukinari Haraoka, Yuki Akieda, Tohru Ishitani, Yoichiro Fujioka, Yusuke Ohba, Sohei Yamada, Yoichiroh Hosokawa, Yusuke Toyama, Takaaki Matsui, Yasuyuki Fujita

  Current biology : CB 30 (4) 670 - 681 2020/02/24 [Refereed][Not invited]
   

  When oncogenic transformation or apoptosis occurs within epithelia, the harmful or dead cells are apically extruded from tissues to maintain epithelial homeostasis. However, the underlying molecular mechanism still remains elusive. In this study, we first show, using mammalian cultured epithelial cells and zebrafish embryos, that prior to apical extrusion of RasV12-transformed cells, calcium wave occurs from the transformed cell and propagates across the surrounding cells. The calcium wave then triggers and facilitates the process of extrusion. IP3 receptor, gap junction, and mechanosensitive calcium channel TRPC1 are involved in calcium wave. Calcium wave induces the polarized movement of the surrounding cells toward the extruding transformed cells. Furthermore, calcium wave facilitates apical extrusion, at least partly, by inducing actin rearrangement in the surrounding cells. Moreover, comparable calcium propagation also promotes apical extrusion of apoptotic cells. Thus, calcium wave is an evolutionarily conserved, general regulatory mechanism of cell extrusion.
• An antiviral drug screening platform with a FRET biosensor for measurement of arenavirus Z assembly

  Tatsuaki Mizutani, Yusuke Ohba, Satoshi Mizuta, Jiro Yasuda, Shuzo Urata

  Cell Structure and Function 0386-7196 2020
• Folding Latency of Fluorescent Proteins Affects the Mitochondrial Localization of Fusion Proteins.

  Sayaka Kashiwagi, Yoichiro Fujioka, Aya O Satoh, Aiko Yoshida, Mari Fujioka, Prabha Nepal, Atsushi Tsuzuki, Ozora Aoki, Sarad Paudel, Hitoshi Sasajima, Yusuke Ohba

  Cell structure and function 44 (2) 183 - 194 2019/12/26 [Refereed][Not invited]
   

  The discovery of fluorescent proteins (FPs) has revolutionized cell biology. The fusion of targeting sequences to FPs enables the investigation of cellular organelles and their dynamics; however, occasionally, such fluorescent fusion proteins (FFPs) exhibit behavior different from that of the native proteins. Here, we constructed a color pallet comprising different organelle markers and found that FFPs targeted to the mitochondria were mislocalized when fused to certain types of FPs. Such FPs included several variants of Aequorea victoria green FP (avGFP) and a monomeric variant of the red FP. Because the FFPs that are mislocalized include FPs with faster maturing or folding mutations, the increase in the maturation rate is likely to prevent their expected localization. Indeed, when we reintroduced amino acid substitutions so that the FP sequences were equivalent to that of wild-type avGFP, FFP localization to the mitochondria was significantly enhanced. Moreover, similar amino acid substitutions improved the localization of mitochondria-targeted pHluorin, which is a pH-sensitive variant of GFP, and its capability to monitor pH changes in the mitochondrial matrix. Our findings demonstrate the importance of selecting FPs that maximize FFP function.Key words: fluorescent protein, organelle, fusion protein, mitochondria.
• Localization of BCR-ABL to Stress Granules Contributes to Its Oncogenic Function.

  Sayaka Kashiwagi, Yoichiro Fujioka, Takeshi Kondo, Aya O Satoh, Aiko Yoshida, Mari Fujioka, Hitoshi Sasajima, Maho Amano, Takanori Teshima, Yusuke Ohba

  Cell structure and function 44 (2) 195 - 204 2019/12/26 [Refereed][Not invited]
   

  The oncogenic tyrosine kinase BCR-ABL activates a variety of signaling pathways and plays a causative role in the pathogenesis of chronic myelogenous leukemia (CML); however, the subcellular distribution of this chimeric protein remains controversial. Here, we report that BCR-ABL is localized to stress granules and that its granular localization contributes to BCR-ABL-dependent leukemogenesis. BCR-ABL-positive granules were not colocalized with any markers for membrane-bound organelles but were colocalized with HSP90a, a component of RNA granules. The number of such granules increased with thapsigargin treatment, confirming that the granules were stress granules. Given that treatment with the ABL kinase inhibitor imatinib and elimination of the N-terminal region of BCR-ABL abolished granule formation, kinase activity and the coiled-coil domain are required for granule formation. Whereas wild-type BCR-ABL rescued the growth defect in IL-3-depleted Ba/F3 cells, mutant BCR-ABL lacking the N-terminal region failed to do so. Moreover, forced tetramerization of the N-terminus-deleted mutant could not restore the growth defect, indicating that granule formation, but not tetramerization, through its N-terminus is critical for BCR-ABL-dependent oncogenicity. Our findings together provide new insights into the pathogenesis of CML by BCR-ABL and open a window for developing novel therapeutic strategies for this disease.Key words: BCR-ABL, subcellular localization, stress granule.
• Clinical efficacy and safety of first-line nilotinib therapy and evaluation of the clinical utility of the FRET-based drug sensitivity test.

  Takeshi Kondo, Mari Fujioka, Shinichi Fujisawa, Kaori Sato, Masumi Tsuda, Takuto Miyagishima, Akio Mori, Hiroshi Iwasaki, Yasutaka Kakinoki, Satoshi Yamamoto, Yoshihito Haseyama, Seisho Ando, Motohiro Shindo, Shuichi Ota, Mitsutoshi Kurosawa, Yusuke Ohba, Takanori Teshima

  International journal of hematology 110 (4) 482 - 489 2019/10 [Refereed][Not invited]
   

  Nilotinib is widely used for primary treatment of patients with chronic myelogenous leukemia (CML). We previously reported that use of an FRET-based drug sensitivity test at diagnosis efficiently predicts the response to treatment with imatinib or dasatinib. Here, we conducted a phase-II study to evaluate the efficacy and safety of nilotinib treatment and identify useful biomarkers, including results of the FRET-based drug sensitivity test, for predicting treatment response. Data from 42 patients were used in the analysis. Major molecular response (MMR), MR4, and MR4.5 rates at 12 months were 64.3, 42.9, and 28.6%, respectively. Grade 3/4 non-hematologic adverse events occurred in 11 patients (26.2%). The dose intensity of nilotinib (> 76.44%) and halving time (HT, < 13.312 days) were identified as significant factors for MMR at 12 months. However, when we focused on patients whose dose intensity of nilotinib was > 76.44%, the FRET-based drug sensitivity test became a predictive factor of MR4 achievement at 12 months. Our study reconfirmed the efficacy and safety of nilotinib treatment in CML patients. Moreover, our results suggest that the FRET-based drug sensitivity test is an independent predictor for achievement of MR4 in patients treated with a sufficient dose intensity of nilotinib.
• Development of Immortalized Human Tumor Endothelial Cells from Renal Cancer.

  Nako Maishi, Hiroshi Kikuchi, Masumi Sato, Hiroko Nagao-Kitamoto, Dorcas A Annan, Shogo Baba, Takayuki Hojo, Misa Yanagiya, Yusuke Ohba, Genichiro Ishii, Kenkichi Masutomi, Nobuo Shinohara, Yasuhiro Hida, Kyoko Hida

  International journal of molecular sciences 20 (18) 2019/09/17 [Refereed][Not invited]
   

  Tumor angiogenesis research and antiangiogenic drug development make use of cultured endothelial cells (ECs) including the human microvascular ECs among others. However, it has been reported that tumor ECs (TECs) are different from normal ECs (NECs). To functionally validate antiangiogenic drugs, cultured TECs are indispensable tools, but are not commercially available. Primary human TECs are available only in small quantities from surgical specimens and have a short life span in vitro due to their cellular senescence. We established immortalized human TECs (h-imTECs) and their normal counterparts (h-imNECs) by infection with lentivirus producing simian virus 40 large T antigen and human telomerase reverse transcriptase to overcome the replication barriers. These ECs exhibited an extended life span and retained their characteristic endothelial morphology, expression of endothelial marker, and ability of tube formation. Furthermore, h-imTECs showed their specific characteristics as TECs, such as increased proliferation and upregulation of TEC markers. Treatment with bevacizumab, an antiangiogenic drug, dramatically decreased h-imTEC survival, whereas the same treatment failed to alter immortalized NEC survival. Hence, these h-imTECs could be a valuable tool for drug screening to develop novel therapeutic agents specific to TECs or functional biological assays in tumor angiogenesis research.
• A Peptide Derived from Phosphoinositide 3-kinase Inhibits Endocytosis and Influenza Virus Infection.

  Yoichiro Fujioka, Aya O Satoh, Kosui Horiuchi, Mari Fujioka, Kaori Tsutsumi, Junko Sasaki, Prabha Nepal, Sayaka Kashiwagi, Sarad Paudel, Shinya Nishide, Asuka Nanbo, Takehiko Sasaki, Yusuke Ohba

  Cell structure and function 44 (1) 61 - 74 2019/04/25 [Refereed][Not invited]
   

  Endocytosis mediates the internalization and ingestion of a variety of endogenous or exogenous substances, including virus particles, under the control of intracellular signaling pathways. We have previously reported that the complex formed between the small GTPase Ras and phosphoinositide 3-kinase (PI3K) translocates from the plasma membrane to endosomes, signaling from which thereby regulates clathrin-independent endocytosis, endosome maturation, influenza virus internalization, and infection. However, the molecular mechanism by which the Ras-PI3K complex is recruited to endosomes remains unclear. Here, we have identified the amino acid sequence responsible for endosomal localization of the Ras-PI3K complex. PI3K lacking this sequence failed to translocate to endosomes, and expression of the peptide comprising this PI3K-derived sequence inhibited clathrin-independent endocytosis, influenza virus internalization, and infection. Moreover, treatment of cells with this peptide in an arginine-rich, cell-penetrating form successfully suppressed influenza virus infection in vitro and ex vivo, making this peptide a potential therapeutic agent against influenza virus infection.Key words: signal transduction, endocytosis, endosome, imaging, influenza virus.
• Antibody-free digital influenza virus counting based on neuraminidase activity.

  Kazuhito V Tabata, Yoshihiro Minagawa, Yuko Kawaguchi, Mana Ono, Yoshiki Moriizumi, Seiya Yamayoshi, Yoichiro Fujioka, Yusuke Ohba, Yoshihiro Kawaoka, Hiroyuki Noji

  Scientific reports 9 (1) 1067 - 1067 2019/01/31 [Refereed][Not invited]
   

  There is large demand for a quantitative method for rapid and ultra-sensitive detection of the influenza virus. Here, we established a digital influenza virus counting (DIViC) method that can detect a single virion without antibody. In the assay, a virion is stochastically entrapped inside a femtoliter reactor array device for the fluorogenic assay of neuraminidase, and incubated for minutes. By analyzing 600,000 reactors, the practical limit of detection reached the order of 103 (PFU)/mL, only 10-times less sensitive than RT-PCR and more than 1000-times sensitive than commercial rapid test kits (RIDTs). Interestingly, neuraminidase activity differed among virions. The coefficient of variance was 30-40%, evidently broader than that of alkaline phosphatase measured as a model enzyme for comparison, suggesting the heterogeneity in size and integrity among influenza virus particles. Sensitivity to oseltamivir also differed between virions. We also tested DIViC using clinical gargle samples that imposes less burden for sampling while with less virus titre. The comparison with RIDTs showed that DIViC was largely superior to RIDTs in the sensitivity with the clinical samples although a few false-positive signals were observed in some clinical samples that remains as a technical challenge.
• Budding of Ebola Virus Particles Requires the Rab11-Dependent Endocytic Recycling Pathway.

  Asuka Nanbo, Yusuke Ohba

  The Journal of infectious diseases 218 (suppl_5) S388-S396  2018/11/22 [Refereed][Not invited]
   

  The Ebola virus-encoded major matrix protein VP40 traffics to the plasma membrane, which leads to the formation of filamentous viral particles and subsequent viral egress. However, the cellular machineries underlying this process are not fully understood. In the present study, we have assessed the role of host endocytic recycling in Ebola virus particle formation. We found that a small GTPase Rab11, which regulates recycling of molecules among the trans-Golgi network, recycling endosomes, and the plasma membrane, was incorporated in Ebola virus-like particles. Although Rab11 predominantly localized in the perinuclear region, it distributed diffusely in the cytoplasm and partly localized in the periphery of the cells transiently expressing VP40. In contrast, Rab11 exhibited a perinuclear distribution when 2 VP40 derivatives that lack ability to traffic to the plasma membrane were expressed. Finally, expression of a dominant-negative form of Rab11 or knockdown of Rab11 inhibited both VP40-induced clusters at the plasma membrane and release of viral-like particles. Taken together, our findings demonstrate that Ebola virus exploits host endocytic recycling machinery to facilitate the trafficking of VP40 to the cell surface and the subsequent release of viral-like particles for its establishment of efficient viral egress.
• ダーモスコープを用いた術後植皮片の観察

  北村 真也, 柳 輝希, 高島 有香, 今福 恵輔, 秦 洋郎, 清水 宏, 藤岡 容一朗, 大場 雄介

  日本皮膚科学会雑誌 (公社)日本皮膚科学会 128 (12) 2665 - 2665 0021-499X 2018/11
• Infection of Epstein⁻Barr Virus in Type III Latency Modulates Biogenesis of Exosomes and the Expression Profile of Exosomal miRNAs in the Burkitt Lymphoma Mutu Cell Lines.

  Asuka Nanbo, Harutaka Katano, Michiyo Kataoka, Shiho Hoshina, Tsuyoshi Sekizuka, Makoto Kuroda, Yusuke Ohba

  Cancers 10 (7) 2018/07/19 [Refereed][Not invited]
   

  Infection of Epstein⁻Barr virus (EBV), a ubiquitous human gamma herpesvirus, is associated with various malignancies in B lymphocytes and epithelial cells. EBV encodes 49 microRNAs in two separated regions, termed the BART and BHRF1 loci. Although accumulating evidence demonstrates that EBV infection regulates the profile of microRNAs in the cells, little is known about the microRNAs in exosomes released from infected cells. Here, we characterized the expression profile of intracellular and exosomal microRNAs in EBV-negative, and two related EBV-infected Burkitt lymphoma cell lines having type I and type III latency by next-generation sequencing. We found that the biogenesis of exosomes is upregulated in type III latently infected cells compared with EBV-negative and type I latently infected cells. We also observed that viral and several specific host microRNAs were predominantly incorporated in the exosomes released from the cells in type III latency. We confirmed that multiple viral microRNAs were transferred to the epithelial cells cocultured with EBV-infected B cells. Our findings indicate that EBV infection, in particular in type III latency, modulates the biogenesis of exosomes and the profile of exosomal microRNAs, potentially contributing to phenotypic changes in cells receiving these exosomes.
• Pretreatment evaluation of fluorescence resonance energy transfer-based drug sensitivity test for patients with chronic myelogenous leukemia treated with dasatinib.

  Takeshi Kondo, Mari Fujioka, Masumi Tsuda, Kazunori Murai, Kohei Yamaguchi, Takuto Miyagishima, Motohiro Shindo, Takahiro Nagashima, Kentaro Wakasa, Nozomu Fujimoto, Satoshi Yamamoto, Masakatsu Yonezumi, Souichi Saito, Shinji Sato, Kazuei Ogawa, Takaaki Chou, Reiko Watanabe, Yuichi Kato, Shuichiro Takahashi, Yoshiaki Okano, Joji Yamamoto, Masatsugu Ohta, Hiroaki Iijima, Koji Oba, Satoshi Kishino, Junichi Sakamoto, Yoji Ishida, Yusuke Ohba, Takanori Teshima

  Cancer science 109 (7) 2256 - 2265 1347-9032 2018/07 [Refereed][Not invited]
   

  Tyrosine kinase inhibitors (TKI) are used for primary therapy in patients with newly diagnosed CML. However, a reliable method for optimal selection of a TKI from the viewpoint of drug sensitivity of CML cells has not been established. We have developed a FRET-based drug sensitivity test in which a CrkL-derived fluorescent biosensor efficiently quantifies the kinase activity of BCR-ABL of living cells and sensitively evaluates the inhibitory activity of a TKI against BCR-ABL. Here, we validated the utility of the FRET-based drug sensitivity test carried out at diagnosis for predicting the molecular efficacy. Sixty-two patients with newly diagnosed chronic phase CML were enrolled in this study and treated with dasatinib. Bone marrow cells at diagnosis were subjected to FRET analysis. The ΔFRET value was calculated by subtraction of FRET efficiency in the presence of dasatinib from that in the absence of dasatinib. Treatment response was evaluated every 3 months by the BCR-ABL1 International Scale. Based on the ΔFRET value and molecular response, a threshold of the ΔFRET value in the top 10% of FRET efficiency was set to 0.31. Patients with ΔFRET value ≥0.31 had significantly superior molecular responses (MMR at 6 and 9 months and both MR4 and MR4.5 at 6, 9, and 12 months) compared with the responses in patients with ΔFRET value <0.31. These results suggest that the FRET-based drug sensitivity test at diagnosis can predict early and deep molecular responses. This study is registered with UMIN Clinical Trials Registry (UMIN000006358).
• A Sialylated Voltage-Dependent Ca2+ Channel Binds Hemagglutinin and Mediates Influenza A Virus Entry into Mammalian Cells.

  Yoichiro Fujioka, Shinya Nishide, Toyoyuki Ose, Tadaki Suzuki, Izumi Kato, Hideo Fukuhara, Mari Fujioka, Kosui Horiuchi, Aya O Satoh, Prabha Nepal, Sayaka Kashiwagi, Jing Wang, Mika Horiguchi, Yuko Sato, Sarad Paudel, Asuka Nanbo, Tadaaki Miyazaki, Hideki Hasegawa, Katsumi Maenaka, Yusuke Ohba

  Cell host & microbe 23 (6) 809 - 818 1931-3128 2018/06/13 [Refereed][Not invited]
   

  Influenza A virus (IAV) infection is initiated by the attachment of the viral glycoprotein hemagglutinin (HA) to sialic acid on the host cell surface. However, the sialic acid-containing receptor crucial for IAV infection has remained unidentified. Here, we show that HA binds to the voltage-dependent Ca2+ channel Cav1.2 to trigger intracellular Ca2+ oscillations and subsequent IAV entry and replication. IAV entry was inhibited by Ca2+ channel blockers (CCBs) or by knockdown of Cav1.2. The CCB diltiazem also inhibited virus replication in vivo. Reintroduction of wild-type but not the glycosylation-deficient mutants of Cav1.2 restored Ca2+ oscillations and virus infection in Cav1.2-depleted cells, demonstrating the significance of Cav1.2 sialylation. Taken together, we identify Cav1.2 as a sialylated host cell surface receptor that binds HA and is critical for IAV entry.
• 体外診断薬開発のための慢性骨髄性白血病におけるBCR-ABL活性測定用改良型FRETバイオセンサー

  大場 雄介, 近藤 健, 豊嶋 崇徳

  臨床薬理の進歩 (公財)臨床薬理研究振興財団 (39) 18 - 26 0914-4366 2018/06 

  アミノ酸置換および核外移行シグナル(NES)の導入により、切断抵抗性が向上した改良型Picklesバイオセンサーを開発したので報告した。フェルスター共鳴エネルギー移動(FRET)に基づくバイオセンサーPickles 2.31のために開発されたオリジナルのプロトコールでは、評価対象細胞をCFPとYFPの蛍光強度比に従って選択する。この基準に従ってPickles 2.31とPickles 2.34 NESの特性を比較したところ、薬剤処理の有無にかかわらず基準を満たす細胞の数が5%から25%増加した。さらに、実際の薬効評価指標であるFRET効率(FRET/CFP蛍光強度比)についても単一細胞レベルで評価した。以前の報告において使用した定義では、FRET効率が2.04より高い細胞は有意に高いBCR-ABL活性を示す。この基準によれば、改良型バイオセンサーはコントロールサンプル中に四つのFRET-high細胞を検出できるが、プロトタイプでは2.04以上の細胞は一つしか検出されなかった。この増加は分析対象細胞数の増加が検出能の向上に寄与した結果と考えられた。
• Dermoscopic evaluation for skin grafts after surgery; neo-vascularization correlates with survival of skin grafts: A prospective study

  Shinya Kitamura, Teruki Yanagi, Yuka Inamura-Takashima, Keisuke Imafuku, Hiroo Hata, Yoichiro Fujioka, Yusuke Ohba, Hiroshi Shimizu

  Journal of Dermatological Science 90 (2) 213 - 216 1873-569X 2018/05/01 [Refereed][Not invited]
  
• miR-23a promotes invasion of glioblastoma via HOXD10-regulated glial-mesenchymal transition.

  Kazuhiro Yachi, Masumi Tsuda, Shinji Kohsaka, Lei Wang, Yoshitaka Oda, Satoshi Tanikawa, Yusuke Ohba, Shinya Tanaka

  Signal transduction and targeted therapy 3 33 - 33 2018 [Refereed][Not invited]
   

  Glioblastoma is the most aggressive and invasive brain tumor and has a poor prognosis; elucidating the underlying molecular mechanisms is essential to select molecular targeted therapies. Here, we investigated the effect of microRNAs on the marked invasiveness of glioblastoma. U373 glioblastoma cells were infected with 140 different microRNAs from an OncomiR library, and the effects of the invasion-related microRNAs and targeted molecules were investigated after repeated Matrigel invasion assays. Screening of the OncomiR library identified miR-23a as a key regulator of glioblastoma invasion. In six glioblastoma cell lines, a positive correlation was detected between the expression levels of miR-23a and invasiveness. A luciferase reporter assay demonstrated that homeobox D10 (HOXD10) was a miR-23a-target molecule, which was verified by high scores from both the PicTar and miRanda algorithms. Forced expression of miR-23a induced expression of invasion-related molecules, including uPAR, RhoA, and RhoC, and altered expression of glial-mesenchymal transition markers such as Snail, Slug, MMP2, MMP9, MMP14, and E-cadherin; however, these changes in expression levels were reversed by HOXD10 overexpression. Thus, miR-23a significantly promoted invasion of glioblastoma cells with polarized formation of focal adhesions, while exogenous HOXD10 overexpression reversed these phenomena. Here, we identify miR-23a-regulated HOXD10 as a pivotal regulator of invasion in glioblastoma, providing a novel mechanism for the aggressive invasiveness of this tumor and providing insight into potential therapeutic targets.
• Ebola virus requires a host scramblase for externalization of phosphatidylserine on the surface of viral particles.

  Asuka Nanbo, Junki Maruyama, Masaki Imai, Michiko Ujie, Yoichiro Fujioka, Shinya Nishide, Ayato Takada, Yusuke Ohba, Yoshihiro Kawaoka

  PLoS pathogens 14 (1) e1006848  1553-7366 2018/01 [Refereed][Not invited]
   

  Cell surface receptors for phosphatidylserine contribute to the entry of Ebola virus (EBOV) particles, indicating that the presence of phosphatidylserine in the envelope of EBOV is important for the internalization of EBOV particles. Phosphatidylserine is typically distributed in the inner layer of the plasma membrane in normal cells. Progeny virions bud from the plasma membrane of infected cells, suggesting that phosphatidylserine is likely flipped to the outer leaflet of the plasma membrane in infected cells for EBOV virions to acquire it. Currently, the intracellular dynamics of phosphatidylserine during EBOV infection are poorly understood. Here, we explored the role of XK-related protein (Xkr) 8, which is a scramblase responsible for exposure of phosphatidylserine in the plasma membrane of apoptotic cells, to understand its significance in phosphatidylserine-dependent entry of EBOV. We found that Xkr8 and transiently expressed EBOV glycoprotein GP often co-localized in intracellular vesicles and the plasma membrane. We also found that co-expression of GP and viral major matrix protein VP40 promoted incorporation of Xkr8 into ebolavirus-like particles (VLPs) and exposure of phosphatidylserine on their surface, although only a limited amount of phosphatidylserine was exposed on the surface of the cells expressing GP and/or VP40. Downregulating Xkr8 or blocking caspase-mediated Xkr8 activation did not affect VLP production, but they reduced the amount of phosphatidylserine on the VLPs and their uptake in recipient cells. Taken together, our findings indicate that Xkr8 is trafficked to budding sites via GP-containing vesicles, is incorporated into VLPs, and then promote the entry of the released EBOV to cells in a phosphatidylserine-dependent manner.
• Epstein-Barr Virus Acquires Its Final Envelope on Intracellular Compartments With Golgi Markers.

  Asuka Nanbo, Takeshi Noda, Yusuke Ohba

  Frontiers in microbiology 9 454 - 454 2018 [Refereed][Not invited]
   

  Herpesvirus subfamilies typically acquire their final envelope in various cytoplasmic compartments such as the trans-Golgi network (TGN), and endosomes prior to their secretion into the extracellular space. However, the sites for the final envelopment of Epstein-Barr virus (EBV), a ubiquitous human gamma herpesvirus, are poorly understood. Here, we characterized the sites for the final envelopment of EBV in Burkitt's lymphoma cell lines induced into the lytic cycle by crosslinking cell surface IgG. Electron microscopy revealed the various stages of maturation and egress of progeny virions including mature EBV in irregular cytoplasmic vesicles. Immunofluorescence staining showed that gp350/220, the major EBV glycoprotein, and the viral capsid antigen, p18, efficiently colocalized with a cis-Golgi marker, GM130. gp350/220 partly colocalized with the TGN, which was distributed in a fragmented and dispersed pattern in the cells induced into the lytic cycle. In contrast, limited colocalization was observed between gp350/220 and endosomal markers, such as a multi-vesicular bodies marker, CD63, a recycling endosome marker, Rab11, and a regulatory secretion vesicles marker, Rab27a. Finally, we observed that treatment of cells with brefeldin A, an inhibitor of vesicle trafficking between the endoplasmic reticulum and Golgi apparatus, resulted in the perinuclear accumulation of gp350/220 and inhibition of its distribution to the plasma membrane. Brefeldin A also inhibited the release of infectious EBV. Taken together, our findings support a model in which EBV acquires its final envelope in intracellular compartments containing markers of Golgi apparatus, providing new insights into how EBV matures.
• The Role of Transforming Growth Factor β in Cell-to-Cell Contact-Mediated Epstein-Barr Virus Transmission.

  Asuka Nanbo, Makoto Ohashi, Hironori Yoshiyama, Yusuke Ohba

  Frontiers in microbiology 9 984 - 984 2018 [Refereed][Not invited]
   

  Infection of Epstein-Barr virus (EBV), a ubiquitous human gamma herpesvirus, is closely linked to various lymphoid and epithelial malignancies. Previous studies demonstrated that the efficiency of EBV infection in epithelial cells is significantly enhanced by coculturing them with latently infected B cells relative to cell-free infection, suggesting that cell-to-cell contact-mediated viral transmission is the dominant mode of infection by EBV in epithelial cells. However, a detailed mechanism underlying this process has not been fully understood. In the present study, we assessed the role of transforming growth factor β (TGF-β), which is known to induce EBV's lytic cycle by upregulation of EBV's latent-lytic switch BZLF1 gene. We have found that 5 days of cocultivation facilitated cell-to-cell contact-mediated EBV transmission. Replication of EBV was induced in cocultured B cells both with and without a direct cell contact in a time-dependent manner. Treatment of a blocking antibody for TGF-β suppressed both induction of the lytic cycle in cocultured B cells and subsequent viral transmission. Cocultivation with epithelial cells facilitated expression of TGF-β receptors in B cells and increased their susceptibility to TGF-β. Finally, we confirmed the spontaneous secretion of TGF-β from epithelial cells, which was not affected by cell-contact. In contrast, the extracellular microvesicles, exosomes derived from cocultured cells partly contributed to cell-to-cell contact-mediated viral transmission. Taken together, our findings support a role for TGF-β derived from epithelial cells in efficient viral transmission, which fosters induction of the viral lytic cycle in the donor B cells.
• Cell competition with normal epithelial cells promotes apical extrusion of transformed cells through metabolic changes

  Shunsuke Kon, Kojiro Ishibashi, Hiroto Katoh, Sho Kitamoto, Takanobu Shirai, Shinya Tanaka, Mihoko Kajita, Susumu Ishikawa, Hajime Yamauchi, Yuta Yako, Tomoko Kamasaki, Tomohiro Matsumoto, Hirotaka Watanabe, Riku Egami, Ayana Sasaki, Atsuko Nishikawa, Ikumi Kameda, Takeshi Maruyama, Rika Narumi, Tomoko Morita, Yoshiteru Sasaki, Ryosuke Enoki, Sato Honma, Hiromi Imamura, Masanobu Oshima, Tomoyoshi Soga, Jun-ichi Miyazaki, Michael R. Duchen, Jin-Min Nam, Yasuhito Onodera, Shingo Yoshioka, Junichi Kikuta, Masaru Ishii, Masamichi Imajo, Eisuke Nishida, Yoichiro Fujioka, Yusuke Ohba, Toshiro Sato, Yasuyuki Fujita

  NATURE CELL BIOLOGY 19 (5) 530 - + 1465-7392 2017/05 [Refereed][Not invited]
   

  Recent studies have revealed that newly emerging transformed cells are often apically extruded from epithelial tissues. During this process, normal epithelial cells can recognize and actively eliminate transformed cells, a process called epithelial defence against cancer (EDAC). Here, we show that mitochondrial membrane potential is diminished in RasV12-transformed cells when they are surrounded by normal cells. In addition, glucose uptake is elevated, leading to higher lactate production. The mitochondrial dysfunction is driven by upregulation of pyruvate dehydrogenase kinase 4 (PDK4), which positively regulates elimination of RasV12-transformed cells. Furthermore, EDAC from the surrounding normal cells, involving filamin, drives the Warburg-effect-like metabolic alteration. Moreover, using a cell-competition mouse model, we demonstrate that PDK-mediated metabolic changes promote the elimination of RasV12-transformed cells from intestinal epithelia. These data indicate that non-cell-autonomous metabolic modulation is a crucial regulator for cell competition, shedding light on the unexplored events at the initial stage of carcinogenesis.
• Rab5-regulated endocytosis plays a crucial role in apical extrusion of transformed cells

  Sayaka Saitoh, Takeshi Maruyama, Yuta Yako, Mihoko Kajita, Yoichiro Fujioka, Yusuke Ohba, Nobuhiro Kasai, Natsu Sugama, Shunsuke Kon, Susumu Ishikawa, Takashi Hayashi, Tomohiro Yamazaki, Masazumi Tada, Yasuyuki Fujita

  PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 114 (12) E2327 - E2336 0027-8424 2017/03 [Refereed][Not invited]
   

  Newly emerging transformed cells are often eliminated from epithelial tissues. Recent studies have revealed that this cancer-preventive process involves the interaction with the surrounding normal epithelial cells; however, the molecular mechanisms underlying this phenomenon remain largely unknown. In this study, using mammalian cell culture and zebrafish embryo systems, we have elucidated the functional involvement of endocytosis in the elimination of RasV12-transformed cells. First, we show that Rab5, a crucial regulator of endocytosis, is accumulated in RasV12-transformed cells that are surrounded by normal epithelial cells, which is accompanied by up-regulation of clathrin-dependent endocytosis. Addition of chlorpromazine or coexpression of a dominant-negative mutant of Rab5 suppresses apical extrusion of RasV12 cells from the epithelium. We also show in zebrafish embryos that Rab5 plays an important role in the elimination of transformed cells from the enveloping layer epithelium. In addition, Rab5-mediated endocytosis of E-cadherin is enhanced at the boundary between normal and RasV12 cells. Rab5 functions upstream of epithelial protein lost in neoplasm (EPLIN), which plays a positive role in apical extrusion of RasV12 cells by regulating protein kinase A. Furthermore, we have revealed that epithelial defense against cancer (EDAC) from normal epithelial cells substantially impacts on Rab5 accumulation in the neighboring transformed cells. This report demonstrates that Rab5-mediated endocytosis is a crucial regulator for the competitive interaction between normal and transformed epithelial cells in mammals.
• Leukemogenic kinase FIP1L1-PDGFRA and a small ubiquitin-like modifier E3 ligase, PIAS1, form a positive cross-talk through their enzymatic activities

  Makoto Ibata, Junko Iwasaki, Yoichiro Fujioka, Koji Nakagawa, Stephanie Darmanin, Masahiro Onozawa, Daigo Hashimoto, Yusuke Ohba, Shigetsugu Hatakeyama, Takanori Teshima, Takeshi Kondo

  CANCER SCIENCE 108 (2) 200 - 207 1347-9032 2017/02 [Refereed][Not invited]
   

  Fusion tyrosine kinases play a crucial role in the development of hematological malignancies. FIP1L1-PDGFRA is a leukemogenic fusion kinase that causes chronic eosinophilic leukemia. As a constitutively active kinase, FIP1L1-PDGFRA stimulates downstream signaling molecules, leading to cellular proliferation and the generation of an anti-apoptotic state. Contribution of the N-terminal FIP1L1 portion is necessary for FIP1L1-PDGFRA to exert its full transforming activity, but the underlying mechanisms have not been fully characterized. We identified PIAS1 as a FIP1L1-PDGFRA association molecule by yeast two-hybrid screening. Our analyses indicate that the FIP1L1 portion of FIP1L1-PDGFRA is required for efficient association with PIAS1. As a consequence of the association, FIP1L1-PDGFRA phosphorylates PIAS1. Moreover, the kinase activity of FIP1L1-PDGFRA stabilizes PIAS1. Therefore, PIAS1 is one of the downstream targets of FIP1L1-PDGFRA. Moreover, we found that PIAS1, as a SUMO E3 ligase, sumoylates and stabilizes FIP1L1-PDGFRA. In addition, suppression of PIAS1 activity by a knockdown experiment resulted in destabilization of FIP1L1-PDGFRA. Therefore, FIP1L1-PDGFRA and PIAS1 form a positive cross-talk through their enzymatic activities. Suppression of sumoylation by ginkgolic acid, a small molecule compound inhibiting a SUMO E1-activating enzyme, also destabilizes FIP1L1-PDGFRA, and while the tyrosine kinase inhibitor imatinib suppresses FIP1L1-PDGFRA-dependent cell growth, ginkgolic acid or siRNA of PIAS1 has a synergistic effect with imatinib. In conclusion, our results suggest that sumoylation by PIAS1 is a potential target in the treatment of FIP1L1-PDGFRA-positive chronic eosinophilic leukemia.
• Improved FRET Biosensor for the Measurement of BCR-ABL Activity in Chronic Myeloid Leukemia Cells

  Mika Horiguchi, Mari Fujioka, Takeshi Kondo, Yoichiro Fujioka, Xinxin Li, Kosui Horiuchi, Aya O. Satoh, Prabha Nepal, Shinya Nishide, Asuka Nanbo, Takanori Teshima, Yusuke Ohba

  CELL STRUCTURE AND FUNCTION 42 (1) 15 - 26 0386-7196 2017 [Refereed][Not invited]
   

  Although the co-development of companion diagnostics with molecular targeted drugs is desirable, truly efficient diagnostics are limited to diseases in which chromosomal translocations or overt mutations are clearly correlated with drug efficacy. Moreover, even for such diseases, few methods are available to predict whether drug administration is effective for each individual patient whose disease is expected to respond to the drug(s). We have previously developed a biosensor based on the principle of Forster resonance energy transfer to measure the activity of the tyrosine kinase BCR-ABL and its response to drug treatment in patient-derived chronic myeloid leukemia cells. The biosensor harbors CrkL, one of the major substrates of BCR-ABL, and is therefore named Pickles after phosphorylation indicator of CrkL en substrate. The efficacy of this technique as a clinical test has been demonstrated, but the number of cells available for analysis is limited in a case-dependent manner, owing to the cleavage of the biosensor in patient-derived leukemia cells. Here, we describe an improved biosensor with an amino acid substitution and a nuclear export signal being introduced. Of the two predicted cleavage positions in CrkL, the mutations inhibited one cleavage completely and the other cleavage partially, thus collectively increasing the number of cells available for drug evaluation. This improved version of the biosensor holds promise in the future development of companion diagnostics to predict responses to tyrosine kinase inhibitors in patients with chronic myeloid leukemia.
• SH2 domain-based FRET biosensor for measuring BCR-ABL activity in living CML cells

  Mari Fujioka, Yumi Asano, Shigeyuki Nakada, Yusuke Ohba

  Methods in Molecular Biology 1555 513 - 534 1064-3745 2017 [Refereed][Not invited]
   

  Fluorescent proteins (FPs) displaying distinct spectra have shed their light on a wide range of biological functions. Moreover, sophisticated biosensors engineered to contain single or multiple FPs, including Förster resonance energy transfer (FRET)-based biosensors, spatiotemporally reveal the molecular mechanisms underlying a variety of pathophysiological processes. However, their usefulness for applied life sciences has yet to be fully explored. Recently, our research group has begun to expand the potential of FPs from basic biological research to the clinic. Here, we describe a method to evaluate the responsiveness of leukemia cells from patients to tyrosine kinase inhibitors using a biosensor based on FP technology and the principle of FRET. Upon phosphorylation of the tyrosine residue of the biosensor, binding of the SH2 domain to phosphotyrosine induces conformational change of the biosensor and brings the donor and acceptor FPs into close proximity. Therefore, kinase activity and response to kinase inhibitors can be monitored by an increase and a decrease in FRET efficiency, respectively. As in basic research, this biosensor resolves hitherto arduous tasks and may provide innovative technological advances in clinical laboratory examinations. State-of-the-art detection devices that enable such innovation are also introduced.
• Fluorescence bioimaging of intracellular signaling and its clinical application

  Ohba Yusuke, Fujioka Yoichiro

  Journal of Oral Biosciences 58 (4) 113 - 119 1880-3865 2016/11 [Refereed][Not invited]
  
• Epstein-Barr virus exploits host endocytic machinery for cell-to-cell viral transmission rather than a virological synapse

  Asuka Nanbo, Kunihiro Kachi, Hironori Yoshiyama, Yusuke Ohba

  JOURNAL OF GENERAL VIROLOGY 97 (11) 2989 - 3006 0022-1317 2016/11 [Refereed][Not invited]
   

  Epstein-Barr virus (EBV) establishes a lifelong latent infection in B lymphocytes and often is found in epithelial cells. Several lines of evidence indicate that viral transmission mediated by cell-to-cell contact is the dominant mode of infection by EBV for epithelial cells. However, its detailed molecular mechanism has not been fully elucidated. We investigated the role of host membrane trafficking machinery in this process. We have found that adhesion molecules critical for this process are expressed in EBV-positive and -negative Burkitt's lymphoma (BL) cells and multiple epithelial cell lines. Treatment with blocking antibodies against beta 1 and beta 2 integrin families and their ligands suppressed EBV transmission in a dose-dependent manner. We also confirmed that adhesion molecules are upregulated in co-cultured BL cells. Immunofluorescence staining revealed that the intracellular adhesion molecule 1 (ICAM-1) distributed to the cell surface and partially co-localized with recycling endosomes in co-cultured BL cells. Moreover, cell-to-cell EBV transmission was inhibited upon blocking endocytic recycling by expression of a dominant-negative form of a small GTPase Rab11 or by knockdown of Rab11, supporting the notion that the endocytic pathway-dependent trafficking of ICAM-1 to the cell surface of BL cells contributes to viral transmission by stabilizing cell-to-cell contact between the donor cells and recipient cells. Finally, we demonstrated that co-cultivation upregulated clathrin-mediated endocytosis in the recipient cells, allowing EBV to be internalized. Taken together, our findings demonstrate that EBV exploits host endocytic machinery in both donor and recipient cells, a process which is facilitated by cell-to-cell contact, thereby promoting successful viral transmission.
• Tumour endothelial cells in high metastatic tumours promote metastasis via epigenetic dysregulation of biglycan

  Nako Maishi, Yusuke Ohba, Kosuke Akiyama, Noritaka Ohga, Jun-ichi Hamada, Hiroko Nagao-Kitamoto, Mohammad Towfik Alam, Kazuyuki Yamamoto, Taisuke Kawamoto, Nobuo Inoue, Akinobu Taketomi, Masanobu Shindoh, Yasuhiro Hida, Kyoko Hida

  SCIENTIFIC REPORTS 6 28039  2045-2322 2016/06 [Refereed][Not invited]
   

  Tumour blood vessels are gateways for distant metastasis. Recent studies have revealed that tumour endothelial cells (TECs) demonstrate distinct phenotypes from their normal counterparts. We have demonstrated that features of TECs are different depending on tumour malignancy, suggesting that TECs communicate with surrounding tumour cells. However, the contribution of TECs to metastasis has not been elucidated. Here, we show that TECs actively promote tumour metastasis through a bidirectional interaction between tumour cells and TECs. Co-implantation of TECs isolated from highly metastatic tumours accelerated lung metastases of low metastatic tumours. Biglycan, a small leucine-rich repeat proteoglycan secreted from TECs, activated tumour cell migration via nuclear factor-kappa B and extracellular signal-regulated kinase 1/2. Biglycan expression was upregulated by DNA demethylation in TECs. Collectively, our results demonstrate that TECs are altered in their microenvironment and, in turn, instigate tumour cells to metastasize, which is a novel mechanism for tumour metastasis.
• Lypd8 promotes the segregation of flagellated microbiota and colonic epithelia

  Ryu Okumura, Takashi Kurakawa, Takashi Nakano, Hisako Kayama, Makoto Kinoshita, Daisuke Motooka, Kazuyoshi Gotoh, Taishi Kimura, Naganori Kamiyama, Takashi Kusu, Yoshiyasu Ueda, Hong Wu, Hideki Iijima, Soumik Barman, Hideki Osawa, Hiroshi Matsuno, Junichi Nishimura, Yusuke Ohba, Shota Nakamura, Tetsuya Iida, Masahiro Yamamoto, Eiji Umemoto, Koichi Sano, Kiyoshi Takeda

  NATURE 532 (7597) 117 - + 0028-0836 2016/04 [Refereed][Not invited]
   

  Colonic epithelial cells are covered by thick inner and outer mucus layers(1,2). The inner mucus layer is free of commensal microbiota, which contributes to the maintenance of gut homeostasis(3-6). In the small intestine, molecules critical for prevention of bacterial invasion into epithelia such as Paneth-cell-derived anti-microbial peptides and regenerating islet-derived 3 (RegIII) family proteins have been identified(7-11). Although there are mucus layers providing physical barriers against the large number of microbiota present in the large intestine, the mechanisms that separate bacteria and colonic epithelia are not fully elucidated. Here we show that Ly6/PLAUR domain containing 8 (Lypd8) protein prevents flagellated microbiota invading the colonic epithelia in mice. Lypd8, selectively expressed in epithelial cells at the uppermost layer of the large intestinal gland, was secreted into the lumen and bound flagellated bacteria including Proteus mirabilis. In the absence of Lypd8, bacteria were present in the inner mucus layer and many flagellated bacteria invaded epithelia. Lypd8(-/-) mice were highly sensitive to intestinal inflammation induced by dextran sulfate sodium (DSS). Antibiotic elimination of Gram-negative flagellated bacteria restored the bacterial-free state of the inner mucus layer and ameliorated DSS-induced intestinal inflammation in Lypd8(-/-) mice. Lypd8 bound to flagella and suppressed motility of flagellated bacteria. Thus, Lypd8 mediates segregation of intestinal bacteria and epithelial cells in the colon to preserve intestinal homeostasis.
• Receptor activator of NF-kappa B ligand induces cell adhesion and integrin alpha 2 expression via NF-kappa B in head and neck cancers

  Tamaki Yamada, Masumi Tsuda, Takanori Wagatsuma, Yoichiro Fujioka, Mari Fujioka, Aya O. Satoh, Kosui Horiuchi, Shinya Nishide, Asuka Nanbo, Yasunori Totsuka, Hisashi Haga, Shinya Tanaka, Masanobu Shindoh, Yusuke Ohba

  SCIENTIFIC REPORTS 6 23545  2045-2322 2016/03 [Refereed][Not invited]
   

  Cellular interactions with the extracellular matrix play critical roles in tumor progression. We previously reported that receptor activator of NF-kappa B ligand (RANKL) specifically facilitates head and neck squamous cell carcinoma (HNSCC) progression in vivo. Here, we report a novel role for RANKL in the regulation of cell adhesion. Among the major type I collagen receptors, integrin alpha 2 was significantly upregulated in RANKL-expressing cells, and its knockdown suppressed cell adhesion. The mRNA abundance of integrin alpha 2 positively correlated with that of RANKL in human HNSCC tissues. We also revealed that RANK-NF-kappa B signaling mediated integrin alpha 2 expression in an autocrine/paracrine manner. Interestingly, the amount of active integrin beta 1 on the cell surface was increased in RANKL-expressing cells through the upregulation of integrin alpha 2 and endocytosis. Moreover, the RANK-integrin alpha 2 pathway contributed to RANKL-dependent enhanced survival in a collagen gel and inhibited apoptosis in a xenograft model, demonstrating an important role for RANKL-mediated cell adhesion in three-dimensional environments.
• A role of the sphingosine-1-phosphate (S1P)-S1P receptor 2 pathway in epithelial defense against cancer (EDAC).

  Sayaka Yamamoto, Yuta Yako, Yoichiro Fujioka, Mihoko Kajita, Takeshi Kameyama, Shunsuke Kon, Susumu Ishikawa, Yusuke Ohba, Yusuke Ohno, Akio Kihara, Yasuyuki Fujita

  Molecular biology of the cell 27 (3) 491 - 9 1059-1524 2016/02/01 [Refereed][Not invited]
   

  At the initial step of carcinogenesis, transformation occurs in single cells within epithelia, where the newly emerging transformed cells are surrounded by normal epithelial cells. A recent study revealed that normal epithelial cells have an ability to sense and actively eliminate the neighboring transformed cells, a process named epithelial defense against cancer (EDAC). However, the molecular mechanism of this tumor-suppressive activity is largely unknown. In this study, we investigated a role for the sphingosine-1-phosphate (S1P)-S1P receptor 2 (S1PR2) pathway in EDAC. First, we show that addition of the S1PR2 inhibitor significantly suppresses apical extrusion of RasV12-transformed cells that are surrounded by normal cells. In addition, knockdown of S1PR2 in normal cells induces the same effect, indicating that S1PR2 in the surrounding normal cells plays a positive role in the apical elimination of the transformed cells. Of importance, not endogenous S1P but exogenous S1P is involved in this process. By using FRET analyses, we demonstrate that S1PR2 mediates Rho activation in normal cells neighboring RasV12-transformed cells, thereby promoting accumulation of filamin, a crucial regulator of EDAC. Collectively these data indicate that S1P is a key extrinsic factor that affects the outcome of cell competition between normal and transformed epithelial cells.
• Attenuation of ligand-induced activation of angiotensin II type 1 receptor signaling by the type 2 receptor via protein kinase C

  Takayuki Inuzuka, Yoichiro Fujioka, Masumi Tsuda, Mari Fujioka, Aya O. Satoh, Kosui Horiuchi, Shinya Nishide, Asuka Nanbo, Shinya Tanaka, Yusuke Ohba

  SCIENTIFIC REPORTS 6 21613  2045-2322 2016/02 [Refereed][Not invited]
   

  Angiotensin II (AII) type 2 receptor (AT2R) negatively regulates type 1 receptor (AT1R) signaling. However, the precise molecular mechanism of AT2R-mediated AT1R inhibition remains poorly understood. Here, we characterized the local and functional interaction of AT2R with AT1R. AT2R colocalized and formed a complex with AT1R at the plasma membrane, even in the absence of AII. Upon AII stimulation, the spatial arrangement of the complex was modulated, as confirmed by Forster resonance energy transfer (FRET) analysis, followed by AT2R internalization along with AT1R. AT2R internalization was specifically observed only in the presence of AT1R; AT2R alone could not be internalized. The AT1R-specific inhibitor losartan completely inhibited both the conformational change and the internalization of AT2R with AT1R, whereas the AT2R-specific inhibitor PD123319 partially hindered these phenomena, demonstrating that the activation of both receptors was indispensable for these effects. In addition, treatment with the protein kinase C (PKC) inhibitors inhibited the ligand-dependent accumulation of AT2R but not that of AT1R in the endosomes. A mutation in the putative phosphorylation sites of AT2R also abrogated the co-internalization of ATR2 with AT1R and the inhibitory effect of ATR2 on AT1R. These data suggest that AT2R inhibits ligand-induced AT1R signaling through the PKC-dependent pathway.
• 腫瘍血管内皮細胞はbiglycanの分泌を介してがんの転移を促進する

  間石 奈湖, 大場 雄介, 秋山 廣輔, 大賀 則孝, 浜田 淳一, 北本 宗子[永尾], Mohammad Alam T., 進藤 正信, 樋田 泰浩, 樋田 京子

  日本癌学会総会記事 74回 E - 1030 0546-0476 2015/10 [Refereed][Not invited]
  
• Tyr724 phosphorylation of ELMO1 by Src is involved in cell spreading and migration via Rac1 activation

  Yoshinori Makino, Masumi Tsuda, Yusuke Ohba, Hiroshi Nishihara, Hirofumi Sawa, Kazuo Nagashima, Shinya Tanaka

  CELL COMMUNICATION AND SIGNALING 13 35  1478-811X 2015/07 [Refereed][Not invited]
   

  Background: The complex of Dock180/ELMO1 that functions as a bipartite guanine nucleotide exchange factor for Rac is essential for diverse physiological and pathological processes of cells such as cell migration, phagocytosis, and invasion of cancer cells. Among the Src-family tyrosine kinases (SFKs), it has been reported that Hck directly phosphorylates ELMO1, regulating phagocytosis by promoting activation of Rac1; however, the involvement of other SFKs in ELMO1 phosphorylation has remained unknown. Here, we identified novel tyrosine (Y) residues of ELMO1 phosphorylated by SFKs, and examined the effects on Rac1 activity, cell adhesion, spreading, and cell motility on extracellular matrix (ECM). Results: In this study, we unveiled that Src and Fyn can induce tyrosine phosphorylation of ELMO1 in in vivo and in vitro phosphorylation assays. Mutational analyses identified both Y720 and Y724 residues of ELMO1 as Src-mediated phosphorylation sites, preferentially on Y724. Single substitution of Y724 to Phe abrogated Rac1 activation triggered by Src. To elucidate the biological function of pY724, we established NIH3T3 cells stably expressing wild-type ELMO1 or its Y724F mutant together with Dock180. Among them, Y724-deficient cells exhibited a depletion of Rac1 activity with diminished phosphorylation of ELMO1 even upon the ECM-stimulation. It is noteworthy that NIH3T3 cells with ELMO1 Y724F were strikingly defective to promote cell spreading on fibronectin-coated dish, concomitantly exhibiting immature assemblies of actin stress fibers and focal adhesions. Eventually, ELMO1 Y724F significantly impaired cell migration. Conclusion: These results define that Src-mediated Y724 phosphorylation in ELMO1 plays a critical role for cell spreading via activation of Rac1, leading to promotion of cell migration. As the overexpression and/or hyperactivation of Src have been shown in a wide variety of human cancers, Src-mediated phosphorylation of Y724 in ELMO1 may regulate cancer cell adhesion to the ECM, invasion into surrounding tissues, and subsequent distant metastasis.
• Adaptor protein CRK induces epithelial-mesenchymal transition and metastasis of bladder cancer cells through HGF/c-Met feedback loop.

  Ryuji Matsumoto, Masumi Tsuda, Lei Wang, Nako Maishi, Takashige Abe, Taichi Kimura, Mishie Tanino, Hiroshi Nishihara, Kyoko Hida, Yusuke Ohba, Nobuo Shinohara, Katsuya Nonomura, Shinya Tanaka

  Cancer science 106 (6) 709 - 17 1347-9032 2015/06 [Refereed][Not invited]
   

  We have previously reported that an adaptor protein CRK, including CRK-I and CRK-II, plays essential roles in the malignant potential of various aggressive human cancers, suggesting the validity of targeting CRK in molecular targeted therapy of a wide range of cancers. Nevertheless, the role of CRK in human bladder cancer with marked invasion, characterized by distant metastasis and poor prognosis, remains obscure. In the present study, immunohistochemistry indicated a striking enhancement of CRK-I/-II, but not CRK-like, in human bladder cancer tissues compared to normal urothelium. We established CRK-knockdown bladder cancer cells using 5637 and UM-UC-3, which showed a significant decline in cell migration, invasion, and proliferation. It is noteworthy that an elimination of CRK conferred suppressed phosphorylation of c-Met and the downstream scaffold protein Gab1 in a hepatocyte growth factor-dependent and -independent manner. In epithelial-mesenchymal transition-related molecules, E-cadherin was upregulated by CRK elimination, whereas N-cadherin, vimentin, and Zeb1 were downregulated. A similar effect was observed following treatment with c-Met inhibitor SU11274. Depletion of CRK significantly decreased cell proliferation of 5637 and UM-UC-3, consistent with reduced activity of ERK. An orthotopic xenograft model with bioluminescent imaging revealed that CRK knockdown significantly attenuated not only tumor volume but also the number of circulating tumor cells, resulted in a complete abrogation of metastasis. Taken together, this evidence uncovered essential roles of CRK in invasive bladder cancer through the hepatocyte growth factor/c-Met/CRK feedback loop for epithelial-mesenchymal transition induction. Thus, CRK might be a potent molecular target in bladder cancer, particularly for preventing metastasis, leading to the resolution of clinically longstanding critical issues.
• Molecular Role of RNF43 in Canonical and Noncanonical Wnt Signaling

  Tadasuke Tsukiyama, Akimasa Fukui, Sayuri Terai, Yoichiro Fujioka, Keisuke Shinada, Hidehisa Takahashi, Terry P. Yamaguchi, Yusuke Ohba, Shigetsugu Hatakeyama

  MOLECULAR AND CELLULAR BIOLOGY 35 (11) 2007 - 2023 0270-7306 2015/06 [Refereed][Not invited]
   

  Wnt signaling pathways are tightly regulated by ubiquitination, and dysregulation of these pathways promotes tumorigenesis. It has been reported that the ubiquitin ligase RNF43 plays an important role in frizzled-dependent regulation of the Wnt/beta-catenin pathway. Here, we show that RNF43 suppresses both Wnt/beta-catenin signaling and noncanonical Wnt signaling by distinct mechanisms. The suppression of Wnt/beta-catenin signaling requires interaction between the extracellular protease-associated (PA) domain and the cysteine-rich domain (CRD) of frizzled and the intracellular RING finger domain of RNF43. In contrast, these N-terminal domains of RNF43 are not required for inhibition of noncanonical Wnt signaling, but interaction between the C-terminal cytoplasmic region of RNF43 and the PDZ domain of dishevelled is essential for this suppression. We further show the mechanism by which missense mutations in the extracellular portion of RNF43 identified in patients with tumors activate Wnt/beta-catenin signaling. Missense mutations of RNF43 change their localization from the endosome to the endoplasmic reticulum (ER), resulting in the failure of frizzled-dependent suppression of Wnt/beta-catenin signaling. However, these mutants retain the ability to suppress noncanonical Wnt signaling, probably due to interaction with dishevelled. RNF43 is also one of the potential target genes of Wnt/beta-catenin signaling. Our results reveal the molecular role of RNF43 and provide an insight into tumorigenesis.
• Fluorescent Protein-based Biosensors to Visualize Signal Transduction beneath the Plasma Membrane

  Yoichiro Fujioka, Asuka Nanbo, Shin-ya Nishide, Yusuke Ohba

  ANALYTICAL SCIENCES 31 (4) 267 - 274 0910-6340 2015/04 [Refereed][Not invited]
   

  In response to extracellular stimuli, cells display a variety of behaviors, including proliferation, differentiation, morphological changes and migration. The analysis of the spatiotemporal regulation of signal transduction in living cells is needed for a better understanding of such behaviors, and such investigations have been greatly accelerated by the development of fluorescent protein-based biosensors. Currently, by using these biosensors a range of molecular actions, including lipid metabolism, protein activation, and ion dynamics, can be visualized in living cells. We recently reported that intracellular calcium, with its relevant downstream signaling pathways consisting of the small GTPase Ras and the lipid kinase phoshoinositide-3-kinase (PI3K), can be exploited in an efficient incorporation of influenza A viruses into host cells via endocytosis using a set of biosensors based on fluorescent proteins and the principle of Forster resonance energy transfer. Here, we focus this review on fluorescent protein-based biosensors that have been utilized in our recent research reports.
• Inhibition of Multidrug Transporter in Tumor Endothelial Cells Enhances Antiangiogenic Effects of Low-Dose Metronomic Paclitaxel

  Kosuke Akiyama, Nako Maishi, Noritaka Ohga, Yasuhiro Hida, Yusuke Ohba, Mohammad Towfik Alam, Taisuke Kawamoto, Hitomi Ohmura, Kenji Yamada, Chisaho Torii, Masanobu Shindoh, Kyoko Hida

  AMERICAN JOURNAL OF PATHOLOGY 185 (2) 572 - 580 0002-9440 2015/02 [Refereed][Not invited]
   

  Tumor angiogenesis plays an important role in tumor progression and metastasis. Tumor endothelial cells (TECs) are a therapeutic target of antiangiogenic chemotherapy that was recently developed and is currently being investigated in the clinic with promising results. Low-dose chemotherapy, which is the Long-term administration of relatively Low doses of chemotherapeutic agents, has been proposed for targeting tumor angiogenesis in various types of cancers. ALthough the efficacy of low-dose chemotherapy has been confirmed in several clinical models, some studies show insufficienttherapeutic effect for malignant cancers. As a possible mechanism of the treatment failure, it has been considered that tumor cells may acquire resistance to this therapy. However, drug resistance by TECs may also be due to another mechanism for resistance of tumor cells to low-dose chemotherapy. We reported elsewhere that TECs were resistant to the anticancer drug paclitaxel, which is a mitotic inhibitor, concomitant with P-glycoprotein up-regulation. Verapamil, a P-glycoprotein inhibitor, abrogated TEC resistance in vitro. Herein, we demonstrated that verapamil coadministration enhanced the effects of Low-dose paclitaxel concomitant with inhibiting tumor angiogenesis in a preclinical in vivo mouse melanoma xenograft model. Furthermore, verapamil coadministration reduced lung metastasis. These results suggest that inhibiting P-gLycoprotein in TECs may be a novel strategy for low-dose chemotherapy targeting TECs.
• [The Ras-PI3K signaling is involved in the regulation of endocytosis and virus internalization].

  Fujioka Y, Ohba Y

  Seikagaku. The Journal of Japanese Biochemical Society 87 (1) 91 - 100 0037-1017 2015/02 [Refereed][Not invited]
  
• P18/Stathmin1 is regulated by miR-31 in ovarian cancer in response to taxane.

  Mohamed Kamel Hassan, Hidemichi Watari, Takashi Mitamura, Zainab Mohamed, Sherif F El-Khamisy, Yusuke Ohba, Noriaki Sakuragi

  Oncoscience 2 (3) 294 - 308 2331-4737 2015 [Refereed][Not invited]
   

  MicroRNAs (miRNAs) have been reported to regulate the development of chemoresistance in many tumors. Stathmin 1 (STMN1) is a microtubule-depolymerizing molecule, involved in chemo-response; however, the mechanism of its regulation is unknown. Herein, the immunohistochemical study indicated significant upregulation of the STMN1 in the ovarian cancer tissues defined as resistant tumors compared with those defined as responsive tumors. STMN1 level elevated in the chemoresistant ovarian cancer cells, KF-TX, compared with the parental, KF, ones. Targeting STMN1 by siRNA restored taxane-sensitivity of KF-TX cells. Screening miRNA profiles from KF/KF-TX cellular set followed by bioinformatics-based prediction, revealed that miR-31 could be a possible regulator of STMN1. Down-modulation of miR-31 was verified by quantitative RT-PCR in the cellular set used. Overexpression of miR-31 in KF-TX cells (KF-TX-miR-31) significantly restored chemo-response and reduced STMN1 expression as well. STMN1 reduction-associated cellular characteristics such as enhanced microtubule polymerization and stability, as indicated by acetylated tubulin quantification, confocal visualization, and G2 phase delay, were observed in KF-TX-miR-31 cells, indicating the functional reduction of STMN1. miR-31 suppressed the luciferase activity in reporter construct containing the STMN1 3'-untranslated region (3'-UTR), confirming that miR-31 directly targets STMN1. miR-31 has therapeutic potency when introduced into ovarian cancer, in combination with taxane.
• Calcium signaling mediated influenza virus entry into host cells.

  Y. Fujioka, M. Tsuda, A. Nanbo, T. Hattori, J. Sasaki, T. Sasaki, T. Miyazaki, Y. Ohba

  MOLECULAR BIOLOGY OF THE CELL 25 1059-1524 2014/12 [Refereed][Not invited]
  
• 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 0021-9258 2014/12 [Refereed][Not invited]
   

  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.
• Sustained elevation of Snail promotes glial-mesenchymal transition after irradiation in malignant glioma

  Roshan Mahabir, Mishie Tanino, Aiman Elmansuri, Lei Wang, Taichi Kimura, Tamio Itoh, Yusuke Ohba, Hiroshi Nishihara, Hiroki Shirato, Masumi Tsuda, Shinya Tanaka

  NEURO-ONCOLOGY 16 (5) 671 - 685 1522-8517 2014/05 [Refereed][Not invited]
   

  Ionizing irradiation is an effective treatment for malignant glioma (MG); however, a higher rate of recurrence with more aggressive phenotypes is a vital issue. Although epithelial-mesenchymal transition (EMT) is involved in irradiation-induced cancer progression, the role for such phenotypic transition in MG remains unknown. To investigate the mechanism of irradiation-dependent tumor progression in MG, we performed immunohistochemistry (IHC) and qRT-PCR using primary and recurrent MG specimens, MG cell lines, and primary culture cells of MG. siRNA technique was used for MG cell lines. In 22 cases of clinically recurrent MG, the expression of the mesenchymal markers vimentin and CD44 was found to be increased by IHC. In paired identical MG of 7 patients, the expression of collagen, MMPs, and YKL-40 were also elevated in the recurrent MGs, suggesting the The Cancer Genome Atlas-based mesenchymal subtype. Among EMT regulators, sustained elevation of Snail was observed in MG cells at 21 days after irradiation. Cells exhibited an upregulation of migration, invasion, numbers of focal adhesion, and MMP-2 production, and all of these mesenchymal features were abrogated by Snail knockdown. Intriguingly, phosphorylation of ERK1/2 and GSK-3 were increased after irradiation in a Snail-dependent manner, and TGF- was elevated in both fibroblasts and macrophages but not in MG cells after irradiation. It was noteworthy that irradiated cells also expressed stemness features such as SOX2 expression and tumor-forming potential in vivo. We here propose a novel concept of glial-mesenchymal transition after irradiation in which the sustained Snail expression plays an essential role.
• Histone Deacetylase Inhibitors Sensitize Lung Cancer Cells to Hyperthermia: Involvement of Ku70/SirT-1 in Thermo-Protection

  Mohamed K. Hassan, Hidemichi Watari, Alaa-eldin Salah-eldin, Ahmed S. Sultan, Zainab Mohamed, Yoichiro Fujioka, Yusuke Ohba, Noriaki Sakuragi

  PLOS ONE 9 (4) e94213  1932-6203 2014/04 [Refereed][Not invited]
   

  This study describes the sensitization mechanism to thermal stress by histone deacetylase inhibitors (HDACIs) in lung cancer cells and shows that Ku70, based on its acetylation status, mediates the protection of lung cancer from hyperthermia (42.5 degrees C, 1-6 hrs). Ku70 regulates apoptosis by sequestering pro-apoptotic Bax. However, its role in thermal stress is not fully understood. The findings showed that, pre-treating lung cancer cells with HDACIs, nicotinamide (NM) or Trichostatin A (TsA) or both significantly enhanced hyperthermia-induced Bax-dependent apoptosis in PC-10 cells. We found that hyperthermia induces SirT-1, Sirtuin, upregulation but not HDAC6 or SirT-3, therefore transfection with dominant negative SirT-1 (Y/H) also eliminated the protection and resulted in more cell death by hyperthermia, in H1299 cells through Bax activation. Hyperthermia alone primed lung cancer cells to apoptosis without prominent death. After hyperthermia Bax was upregulated, Bcl-2 was downregulated, the Bax/Bcl-2 ratio was inversed and Bax/Bcl-2 heterodimer was dissociated. Although hyperthermia did not affect total Ku70 expression level, it stimulated Ku70 deacetylation, which in turn could bind more Bax in the PC-10 cells. These findings suggest an escape mechanism from hyperthermia-induced Bax activation. To verify the role of Ku70 in this protection mechanism, Ku70 was silenced by siRNA. Ku70 silencing significantly sensitized the lung cancer cells to hyperthermia. The Ku70 KD cells underwent cytotoxic G1 arrest and caspase-dependant apoptosis when compared to scrambled transfectants which showed only G2/M cytostatic arrest in the cell lines investigated, suggesting an additional cell cycle-dependent, novel, role of Ku70 in protection from hyperthermia. Taken together, our data show a Ku70-dependent protection mechanism from hyperthermia. Targeting Ku70 and/or its acetylation during hyperthermia may represent a promising therapeutic approach for lung cancer.
• Lysosomal Interaction of Akt with Phafin2: A Critical Step in the Induction of Autophagy

  Mami Matsuda-Lennikov, Futoshi Suizu, Noriyuki Hirata, Manabu Hashimoto, Kohki Kimura, Tadashi Nagamine, Yoichiro Fujioka, Yusuke Ohba, Toshihiko Iwanaga, Masayuki Noguchi

  PLOS ONE 9 (1) e79795  1932-6203 2014/01 [Refereed][Not invited]
   

  Autophagy is an evolutionarily conserved mechanism for the gross disposal of intracellular proteins in mammalian cells and dysfunction in this pathway has been associated with human disease. Although the serine threonine kinase Akt is suggested to play a role in this process, little is known about the molecular mechanisms by which Akt induces autophagy. Using a yeast two-hybrid screen, Phafin2 (EAPF or PLEKHF2), a lysosomal protein with a unique structure of N-terminal PH (pleckstrin homology) domain and C-terminal FYVE (Fab 1, YOTB, Vac 1, and EEA1) domain was found to interact with Akt. A sucrose gradient fractionation experiment revealed that both Akt and Phafin2 co-existed in the same lysosome enriched fraction after autophagy induction. Confocal microscopic analysis and BiFC analysis demonstrated that both Akt and Phafin2 accumulate in the lysosome after induction of autophagy. BiFC analysis using PtdIns (3) P interaction defective mutant of Phafin2 demonstrated that lysosomal accumulation of the Akt-Phafin2 complex and subsequent induction of autophagy were lysosomal PtdIns (3) P dependent events. Furthermore, in murine macrophages, both Akt and Phafin2 were required for digestion of fluorescent bacteria and/or LPS-induced autophagy. Taken together, these findings establish that lysosomal accumulation of Akt and Phafin2 is a critical step in the induction of autophagy via an interaction with PtdIns (3)P.
• Apoptosis and Molecular Targeting Therapy in Cancer

  Mohamed Hassan, Hidemichi Watari, Ali AbuAlmaaty, Yusuke Ohba, Noriaki Sakuragi

  BIOMED RESEARCH INTERNATIONAL 2014 150845  2314-6133 2014 [Refereed][Not invited]
   

  Apoptosis is the programmed cell death which maintains the healthy survival/death balance in metazoan cells. Defect in apoptosis can cause cancer or autoimmunity, while enhanced apoptosis may cause degenerative diseases. The apoptotic signals contribute into safeguarding the genomic integrity while defective apoptosis may promote carcinogenesis. The apoptotic signals are complicated and they are regulated at several levels. The signals of carcinogenesis modulate the central control points of the apoptotic pathways, including inhibitor of apoptosis (IAP) proteins and FLICE-inhibitory protein (c-FLIP). The tumor cells may use some of several molecular mechanisms to suppress apoptosis and acquire resistance to apoptotic agents, for example, by the expression of antiapoptotic proteins such as Bcl-2 or by the downregulation or mutation of proapoptotic proteins such as BAX. In this review, we provide the main regulatory molecules that govern the main basic mechanisms, extrinsic and intrinsic, of apoptosis in normal cells. We discuss how carcinogenesis could be developed via defective apoptotic pathways or their convergence. We listed some molecules which could be targeted to stimulate apoptosis in different cancers. Together, we briefly discuss the development of some promising cancer treatment strategies which target apoptotic inhibitors including Bcl-2 family proteins, IAPs, and c-FLIP for apoptosis induction.
• Beneficial innate signaling interference for antibacterial responses by a Toll-like receptor-mediated enhancement of the MKP-IRF3 axis

  Hideo Negishi, Kosuke Matsuki, Nobuyasu Endo, Hana Sarashina, Shoji Miki, Atsushi Matsuda, Keiko Fukazawa, Naoko Taguchi-Atarashi, Hiroaki Ikushima, Hideyuki Yanai, Junko Nishio, Kenya Honda, Yoichiro Fujioka, Yusuke Ohba, Tetsuo Noda, Shun'ichiro Taniguchi, Eisuke Nishida, Yongliang Zhang, Hongbo Chi, Richard A. Flavell, Tadatsugu Taniguchi

  PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 110 (49) 19884 - 19889 0027-8424 2013/12 [Refereed][Not invited]
   

  A major function of innate immune receptors is to recognize pathogen-associated molecular patterns and then evoke immune responses appropriate to the nature of the invading pathogen(s). Because innate immune cells express various types of these receptors, distinct combinations of signaling pathways are activated in response to a given pathogen. Although the conventional wisdom is that these signaling pathways cooperate with one another to ensure an effective host response, a more nuanced view recognizes antagonism between the individual pathways, where the attenuation of a signaling pathway(s) by others may shape the immune response. In this study, we show that, on Listeria monocytogenes infection, Toll-like receptor-triggered MyD88 signaling pathways suppress type I IFN gene induction, which is detrimental to macrophage bactericidal activity. These pathways target and suppress the IFN regulatory factor 3 (IRF3) transcription factor that is activated by the stimulator of IFN genes-TANK-binding kinase-1 kinase pathway. We also provide evidence for the involvement of the MAPK phosphatase family members, which renders IRF3 hypophosphorylated on Toll-like receptor signaling by enhancing the formation of an MAPK phosphatase-IRF3-TANK-binding kinase-1 ternary complex. This study, therefore, reveals a hitherto unrecognized and important contribution of a beneficial innate signaling interference against bacterial infections.
• A Ca2+-dependent signalling circuit regulates influenza A virus internalization and infection

  Yoichiro Fujioka, Masumi Tsuda, Asuka Nanbo, Tomoe Hattori, Junko Sasaki, Takehiko Sasaki, Tadaaki Miyazaki, Yusuke Ohba

  NATURE COMMUNICATIONS 4 2763  2041-1723 2013/11 [Refereed][Not invited]
   

  Various viruses enter host cells via endocytosis, but the molecular mechanisms underlying the specific internalization pathways remain unclear. Here we show that influenza A viruses (IAVs) enter cells via redundant pathways of clathrin-mediated and clathrin-independent endocytosis, with intracellular Ca2+ having a central role in regulation of both pathways by activating a signalling axis comprising RhoA, Rho-kinase, phosphatidylinositol 4-phosphate 5-kinase (PIP5K) and phospholipase C (PLC). IAV infection induces oscillations in the cytosolic Ca2+ concentration of host cells, the prevention of which markedly attenuates virus internalization and infection. The small GTPase RhoA is found both to function downstream of the virus-induced Ca2+ response and itself to induce Ca2+ oscillations in a manner dependent on Rho-kinase and subsequent PIP5K-PLC signalling. This signalling circuit regulates both clathrin-mediated and clathrin-independent endocytosis during virus infection and seems to constitute a key mechanism for regulation of IAV internalization and infection.
• 血管内皮細胞によるがん転移促進(Endothelial cells promote tumor metastasis)

  間石 奈湖, 大場 雄介, 大賀 則孝, 秋山 廣輔, 山本 和幸, 浜田 淳一, 川本 泰輔, アラム・モハメド・トウフィック, 大村 瞳, 進藤 正信, 樋田 泰浩, 樋田 京子

  日本癌学会総会記事 72回 127 - 127 0546-0476 2013/10 [Refereed][Not invited]
  
• P-gp阻害剤はメトロノミックケモセラピーの血管新生阻害効果を強める(P-gp inhibitor enhance antiangiogenic activity in metronomic chemotherapy)

  秋山 廣輔, 大賀 則孝, 樋田 泰浩, 間石 奈湖, ムハンマド・トフィック, 川本 泰輔, 大村 瞳, 山田 健司, 鳥居 ちさほ, 進藤 正信, 大場 雄介, 樋田 京子

  日本癌学会総会記事 72回 127 - 127 0546-0476 2013/10 [Refereed][Not invited]
  
• Inhibition of influenza A virus infection by Galectin-9

  Tomoe Hattori, Tomohiro Arikawa, Yoichiro Fujioka, Junki Maruyama, Yousuke Nakayama, Yusuke Ohba, Toshiro Niki, Tadaaki Miyazaki, Mitsuomi Hirashima, Hiroshi Kida

  JAPANESE JOURNAL OF VETERINARY RESEARCH 61 (1-2) 5 - 18 0047-1917 2013/05 [Refereed][Not invited]
   

  Galectin-9 (Gal-9) inhibited the infection of H1N1, H3N2 and H5N1 influenza A viruses in vitro and in vivo. Fifty percent effective doses (ED50) of Gal-9 were 0.1-0.5 mu M depending on virus strains in the plaque reduction assay. Gal-9 but not Gal-1 bound to the virus particles of A/Puerto Rico/8/34 (H1N1) (PR/8), resulting in inhibition of virus attachment to the host cells. Lactose but not sucrose inhibited the binding of Gal-9 to the viruses. Endogenous Gal-9 expression was detected and increased with the course of infection with influenza A viruses in mice. Fifty percent of Gal-9-transgenic mice survived after the challenge with PR/8, while all of the wild-type mice died. Gal-9 treatment of mice affected diminishing influenza virus replication in the lungs, body weight loss and the expression level of inflammatory cytokines. Combined administration of Gal-9 and oseltamivir was more effective than the use of single compound in mouse model. The present results indicate that Gal-9 is a candidate compound for influenza A virus infection therapy.
• All members of the EPI64 subfamily of TBC/RabGAPs also have GAP activities towards Ras

  Hiroyuki Nagai, Sayaka Yasuda, Yusuke Ohba, Mitsunori Fukuda, Takeshi Nakamura

  Journal of Biochemistry 153 (3) 283 - 288 0021-924X 2013/03 [Refereed][Not invited]
   

  The importance of interconnective signalling networks between distinct GTPases and their regulators is being recognized. EPI64C/TBC1D10C/carabin, a haematopoietically enriched GTPase-activating protein (GAP) for Rab35, has been shown to exhibit RasGAP activity. Owing to the diverged Rab specificities among the EPI64 members (EPI64A-C) and the relatively weak sequence conservation between EPI64A/B and EPI64C in their catalytic TBC domains, it is difficult to predict whether EPI64A and B will also have RasGAP activities. Therefore, in this study, we examined the RasGAP activities of all three EPI64 subfamily members. We found that EPI64A-C exhibited in vivo GAP activities towards Ras using three independent methods, spectrofluorometry with Förster resonance energy transfer (FRET) sensors, the Bos' pull-down assay and time-lapse FRET imaging. EPI64A and B were predominantly localized at the periphery of COS-7 cells. In COS-7 cells, confocal FRET imaging showed that H-Ras activity was higher at the Golgi than at the plasma membrane. Thus, we propose that EPI64A and B, which are ubiquitously expressed members of the EPI64 subfamily, inactivate Ras and certain Rabs at the periphery of cells. © 2012 The Authors 2012.
• Fluorescent protein-based biosensors and their clinical applications

  Yusuke Ohba, Yoichiro Fujioka, Shigeyuki Nakada, Masumi Tsuda

  Progress in Molecular Biology and Translational Science 113 313 - 348 1877-1173 2013 [Refereed][Not invited]
   

  Green fluorescent protein and its relatives have shed their light on a wide range of biological problems. To date, with a color palette consisting of fluorescent proteins with different spectra, researchers can "paint" living cells as they desire. Moreover, sophisticated biosensors engineered to contain single or multiple fluorescent proteins, including FRET-based biosensors, spatiotemporally unveil molecular mechanisms underlying physiological processes. Although such molecules have contributed considerably to basic research, their abilities to be used in applied life sciences have yet to be fully explored. Here, we review the molecular bases of fluorescent proteins and fluorescent protein-based biosensors and focus on approaches aimed at applying such proteins to the clinic. © 2013 Elsevier Inc.
• Simultaneous inhibition of Src and Aurora kinases by SU6656 induces therapeutic synergy in human synovial sarcoma growth, invasion and angiogenesis in vivo

  Ryuta Arai, Masumi Tsuda, Takuya Watanabe, Toyoyuki Ose, Chikashi Obuse, Katsumi Maenaka, Akio Minami, Yusuke Ohba

  EUROPEAN JOURNAL OF CANCER 48 (15) 2417 - 2430 0959-8049 2012/10 [Refereed][Not invited]
   

  Synovial sarcoma is an obstinate, high-grade malignancy because of its modest responses to radiotherapy and chemotherapy; the identification of effective therapeutics for this sarcoma is therefore necessary. Inhibition of Src family kinases (SFKs) suppresses the proliferation of synovial sarcoma cells in vitro, as we have previously reported. In this study, to validate the efficacy of Src inhibition in vivo, we employed SU6656, which was originally identified as a specific SFK inhibitor. SU6656 treatment significantly impaired the growth of established, existing tumours formed by synovial sarcoma cells in mice. Tumour cell invasion into the surrounding tissues was also abolished by SU6656. It is noteworthy that SU6656 but not PP2 induced a defect in cleavage furrow formation during cytokinesis, resulting in G2/M accumulation and subsequent apoptosis. Intriguingly, SU6656 abrogated the catalytic activities of Aurora kinases and led to the down-regulation of phosphorylated histone H3 coincidently with p53 accumulation, as did the Aurora kinase inhibitor VX-680. Structural comparison indicated an extensive similarity between the catalytic domains of SFKs and Aurora kinases. The structural analysis also revealed the potential binding mode of SU6656 to the ATP-binding cleft of Aurora B via four hydrogen bonds. SU6656 prevented angiogenesis within the tumours by attenuating vascular endothelial growth factor (VEGF) production by tumour cells and the subsequent chemotaxis of endothelial cells; these effects were the result of the inhibition of SFKs but not Aurora kinases. Based on these results, we hereby report a novel property of SU6656 as a dual inhibitor of SFKs and Aurora kinases, the suppression of both of which effectively abrogates tumour development and the progression of synovial sarcoma in vivo. (C) 2011 Elsevier Ltd. All rights reserved.
• Tumor-infiltrating DCs suppress nucleic acid-mediated innate immune responses through interactions between the receptor TIM-3 and the alarmin HMGB1

  Shigeki Chiba, Muhammad Baghdadi, Hisaya Akiba, Hironori Yoshiyama, Ichiro Kinoshita, Hirotoshi Dosaka-Akita, Yoichiro Fujioka, Yusuke Ohba, Jacob V. Gorman, John D. Colgan, Mitsuomi Hirashima, Toshimitsu Uede, Akinori Takaoka, Hideo Yagita, Masahisa Jinushi

  NATURE IMMUNOLOGY 13 (9) 832 - 842 1529-2908 2012/09 [Refereed][Not invited]
   

  The mechanisms by which tumor microenvironments modulate nucleic acid mediated innate immunity remain unknown. Here we identify the receptor TIM-3 as key in circumventing the stimulatory effects of nucleic acids in tumor immunity. Tumor-associated dendritic cells (DCs) in mouse tumors and patients with cancer had high expression of TIM-3. DC-derived TIM-3 suppressed innate immune responses through the recognition of nucleic acids by Toll-like receptors and cytosolic sensors via a galectin-9-independent mechanism. In contrast, TIM-3 interacted with the alarmin HMGB1 to interfere with the recruitment of nucleic acids into DC endosomes and attenuated the therapeutic efficacy of DNA vaccination and chemotherapy by diminishing the immunogenicity of nucleic acids released from dying tumor cells. Our findings define a mechanism whereby tumor microenvironments suppress antitumor immunity mediated by nucleic acids.
• がん転移における腫瘍血管内皮細胞の役割(The role of tumor endothelial cells in tumor metastasis)

  間石 奈湖, 大場 雄介, 大賀 則孝, 秋山 廣輔, 山本 和幸, 浜田 淳一, 川本 泰輔, 大澤 崇宏, 近藤 美弥子, 大村 瞳, 進藤 正信, 樋田 泰浩, 樋田 京子

  日本癌学会総会記事 71回 85 - 85 0546-0476 2012/08 [Refereed][Not invited]
  
• 細胞内シグナル伝達の可視化技術と分子標的治療薬の耐性判定への応用

  大場雄介, 津田真寿美

  生化学 日本生化学会 84 (5) 359 - 365 0037-1017 2012/05/25 [Not refereed][Not invited]
  
• Biosensors for BCR-ABL activity and their application to cancer

  Yusuke Ohba, Stephanie Darmanin, Tatsuaki Mizutani, Masumi Tsuda, Takeshi Kondo

  Biosensors and Cancer 268 - 283 2012/01/01 

  The emergence of imatinib mesylate designed to inhibit the causativeprotein of chronic myeloid leukemia-BCR-ABL, has radicallyinnovated the treatment of this disease, making it now controllable byoral drugs. However resistance and intolerance are still of concern in asubstantial number of patients. A recently developed biosensor, whichutilizes the major BCR-ABL substrate CrkL, green fluorescent proteintechnology, and the principle of Förster resonance energy transfer, to overcome these issues in chronic myeloid leukemia treatment isintroduced here. This novel diagnostic method for the measurementof BCR-ABL activity has a higher sensitivity than that of establishedtechniques. It can be used as an accurate gauge of BCR-ABL kinaseactivity in small numbers of living cells, and is a useful tool for thedetection of minor drug-resistant populations, as well as the predictionof the clinical course after drug treatment and future onset of drugresistance using patient cells. In consideration of its quick and practicalnature, this method is potentially a promising tool for the predictionof both current and future therapeutic responses in individual patientswith chronic myeloid leukemia, which will surely be beneficial for bothpatients and clinicians. Moreover, the biosensor now provides a newwindow for the application of fluorescent proteins in the practical sceneof clinical medicine, whereas to date, their contributions have only beenlimited to basic research fields.
• Visualization of intracellular signaling and its application to assessment of response to molecular target drugs

  Yusuke Ohba, Masumi Tsuda

  Seikagaku 84 (5) 359 - 365 0037-1017 2012 [Refereed][Not invited]
  
• FRETバイオセンサーによるCML細胞のチロシンキナーゼ阻害薬感受性試験~臨床検体での有用性の検討

  近藤健, 金安顕子, 盛暁生, 入江達朗, 津田真寿美, 森岡正信, 今村雅寛, 大場雄介

  臨床血液 52 (9) 1133  0485-1439 2011/09/30 [Not refereed][Not invited]
  
• DJ-1 associates with synaptic membranes

  Yukiko Usami, Taku Hatano, Satoshi Imai, Shin-ichiro Kubo, Shigeto Sato, Shinji Saiki, Yoichiro Fujioka, Yusuke Ohba, Fumiaki Sato, Manabu Funayama, Hiroto Eguchi, Kaori Shiba, Hiroyoshi Ariga, Jie Shen, Nobutaka Hattori

  NEUROBIOLOGY OF DISEASE 43 (3) 651 - 662 0969-9961 2011/09 [Refereed][Not invited]
   

  Parkinson's disease (PD) is a neurodegenerative disorder caused by loss of dopaminergic neurons. Although many reports have suggested that genetic factors are implicated in the pathogenesis of PD, molecular mechanisms underlying selective dopaminergic neuronal degeneration remain unknown. DJ-1 is a causative gene for autosomal recessive form of PARK7-linked early-onset PD. A number of studies have demonstrated that exogenous DJ-1 localizes within mitochondria and the cytosol, and functions as a molecular chaperon, as a transcriptional regulator, and as a cell protective factor against oxidative stress. However, the precise subcellular localization and function of endogenous DJ-1 are not well known. The mechanisms by which mutations in DJ-1 contributes to neuronal degeneration also remain poorly understood. Here we show by immunocytochemistry that DJ-1 distributes to the cytosol and membranous structures in a punctate appearance in cultured cells and in primary neurons obtained from mouse brain. Interestingly. DJ-1 colocalizes with the Golgi apparatus proteins GM130 and the synaptic vesicle proteins such as synaptophysin and Rab3A. Forster resonance energy transfer analysis revealed that a small portion of DJ-1 interacts with synaptophysin in living cells. Although the wild-type DJ-1 protein directly associates with membranes without an intermediary protein, the pathogenic L166P mutation of DJ-1 exhibits less binding to synaptic vesicles. These results indicate that DJ-1 associates with membranous organelles including synaptic membranes to exhibit its normal function. (C) 2011 Elsevier Inc. All rights reserved.
• 腫瘍血管内皮細胞とがん転移との相互作用解析(Analysis of interaction between tumor endothelial cells and tumor metastasis)

  間石 奈湖, 大賀 則孝, 樋田 泰浩, 大場 雄介, 浜田 淳一, 秋山 廣輔, 山本 和幸, 大澤 崇宏, 近藤 美弥子, 川本 泰輔, 進藤 正信, 井上 農夫男, 樋田 京子

  日本癌学会総会記事 70回 430 - 430 0546-0476 2011/09 [Refereed][Not invited]
  
• [Visualization of cellular signaling by fluorescent proteins].

  Ohba Y, Tsuda M

  Nihon yakurigaku zasshi. Folia pharmacologica Japonica 1 138 (1) 13 - 17 0015-5691 2011/07 [Refereed][Not invited]
   

  下村脩博士によって，Aequorea victoria の発光器官から緑色蛍光タンパク質GFP（green fluorescent protein）が発見され，1992年にそのcDNAが単離されて以来，生細胞イメージングは生物学研究の必須ツールになっている．GFPはcDNAの細胞導入のみで，生理的環境下での目的タンパク質の局在や局在変化を可視化し，種々のカラーバリアントが入手可能な現在では複数のタンパク質の挙動の同時観察も可能である．また，フェルスター共鳴エネルギー移動（FRET: Förster resonance energy transfer）や蛍光タンパク質再構成法（BiFC: bimolecular fluorescence complementation）等の技術を用いることで，個々のタンパク質の局在や動態のみならずタンパク質の質的変化，つまりタンパク質間相互作用・構造変化等の時間的・空間的な変化の解析も可能である．これらの手法は細胞内シグナル伝達のダイナミクスを解析するために，最も適したツールと言っても過言ではない．本稿では，蛍光イメージングの基礎や応用例の紹介と各実験系が持つ得失を比較し，それぞれの実験系が何を可視化するのに適しているかを議論したい．
• RANKL Expression Specifically Observed in Vivo Promotes Epithelial Mesenchymal Transition and Tumor Progression

  Tamaki Yamada, Masumi Tsuda, Tomomi Takahashi, Yasunori Totsuka, Masanobu Shindoh, Yusuke Ohba

  AMERICAN JOURNAL OF PATHOLOGY 178 (6) 2845 - 2856 0002-9440 2011/06 [Refereed][Not invited]
   

  Recent findings have focused attention on the molecular consequences of the microenvironment in tumor progression, but events occurring in cancer cells themselves in response to their ambient conditions remain obscure. Here, we identify receptor activator of nuclear factor kappa B ligand (RANKL) as a microenvironment-specific factor essential for tumorigenesis in vivo, using head and neck squamous cell carcinoma (HNSCC) as a model. In human HNSCC tissues, RANKL is abundantly expressed, and its expression level correlates with the histological grade of differentiation. RANKL levels are significantly higher in poorly differentiated SCCs than in well or moderately differentiated SCCs. In contrast, all HNSCC cell lines tested displayed extremely low RANKL expression; however, RANKL is efficiently up-regulated when these cell lines are inoculated in the head and neck region of mice. RANKL expression is restored in a microenvironment-specific manner, and cannot be observed when the cells are inoculated in the hindlimbs. Forced expression of RANKL compensates for tumor growth in the hindlimb milieu, promotes epithelial mesenchymal transition, and induces tumor angiogenesis, in a manner independent of vascular endothelial growth factor (VEGF). These results implicate RANKL expression causatively in tumor growth and progression in HNSCC in vivo. RANKL may provide a novel functional marker for biological malignancy and a therapeutic target based on the specific nature of the microenvironment. (Am J Pathol 2011, 178:2845-2854. DOI: 10.1016/j.ajpath.2011.02.003)
• IRF3 regulates cardiac fibrosis but not hypertrophy in mice during angiotensin II-induced hypertension

  Kensuke Tsushima, Tomoko Osawa, Hideyuki Yanai, Akira Nakajima, Akinori Takaoka, Ichiro Manabe, Yusuke Ohba, Yasushi Imai, Tadatsugu Taniguchi, Ryozo Nagai

  FASEB JOURNAL 25 (5) 1531 - 1543 0892-6638 2011/05 [Refereed][Not invited]
   

  Hypertension is a typical modern lifestyle-related disease that is closely associated with the development of cardiovascular disorders. Elevation of angiotensin II (ANG II) is one of several critical factors for hypertension and heart failure; however, the mechanisms underlying the ANG II-mediated pathogenesis are still poorly understood. Here, we show that ANG II-mediated cardiac fibrosis, but not hypertrophy, is regulated by interferon regulatory factor 3 (IRF3), which until now has been exclusively studied in the innate immune system. In a ANG II-infusion mouse model (3.0 mg/kg/d), we compared IRF3-deficient mice (Irf3(-/-)/Bcl2l12(-/-)) with matched wild-type (WT) controls. The development of cardiac fibrosis [3.95 +/- 0.62% (WT) vs. 1.41 +/- 0.46% (Irf3(-/-)/Bcl2l12(-/-)); P < 0.01] and accompanied reduction in left ventricle end-diastolic dimension [2.89 +/- 0.10 mm (WT) vs. 3.51 +/- 0.15 mm (Irf3(-/-)/Bcl2l12(-/-)); P=0.012] are strongly suppressed in Irf3(-/-)/Bcl2l12(-/-) mice, whereas hypertrophy still develops. Further, we provide evidence for the activation of IRF3 by ANG II signaling in mouse cardiac fibroblasts. Unlike the activation of IRF3 by innate immune receptors, IRF3 activation by ANG II is unique in that it is activated through the canonical ERK signaling pathway. Thus, our present study reveals a hitherto unrecognized function of IRF3 in cardiac remodeling, providing new insight into the progression of hypertension-induced cardiac pathogenesis.-Tsushima, K., Osawa, T., Yanai, H., Nakajima, A., Takaoka, A., Manabe, I., Ohba, Y., Imai, Y., Taniguchi, T., Nagai, R. IRF3 regulates cardiac fibrosis but not hypertrophy in mice during angiotensin II-induced hypertension. FASEB J. 25, 1531-1543 (2011). www.fasebj.org
• ZAPS is a potent stimulator of signaling mediated by the RNA helicase RIG-I during antiviral responses

  Sumio Hayakawa, Souichi Shiratori, Hiroaki Yamato, Takeshi Kameyama, Chihiro Kitatsuji, Fumi Kashigi, Showhey Goto, Shoichiro Kameoka, Daisuke Fujikura, Taisho Yamada, Tatsuaki Mizutani, Mika Kazumata, Maiko Sato, Junji Tanaka, Masahiro Asaka, Yusuke Ohba, Tadaaki Miyazaki, Masahiro Imamura, Akinori Takaoka

  NATURE IMMUNOLOGY 12 (1) 37 - U56 1529-2908 2011/01 [Not refereed][Not invited]
   

  The poly(ADP-ribose) polymerases (PARPs) participate in many biological and pathological processes. Here we report that the PARP-13 shorter isoform (ZAPS), rather than the full-length protein (ZAP), was selectively induced by 5'-triphosphate-modified RNA (3pRNA) and functioned as a potent stimulator of interferon responses in human cells mediated by the RNA helicase RIG-I. ZAPS associated with RIG-I to promote the oligomerization and ATPase activity of RIG-I, which led to robust activation of IRF3 and NF-kappa B transcription factors. Disruption of the gene encoding ZAPS resulted in impaired induction of interferon-alpha (IFN-alpha), IFN-beta and other cytokines after viral infection. These results indicate that ZAPS is a key regulator of RIG-I signaling during the innate antiviral immune response, which suggests its possible use as a therapeutic target for viral control.
• Induction of Colonic Regulatory T Cells by Indigenous Clostridium Species

  Koji Atarashi, Takeshi Tanoue, Tatsuichiro Shima, Akemi Imaoka, Tomomi Kuwahara, Yoshika Momose, Genhong Cheng, Sho Yamasaki, Takashi Saito, Yusuke Ohba, Tadatsugu Taniguchi, Kiyoshi Takeda, Shohei Hori, Ivaylo I. Ivanov, Yoshinori Umesaki, Kikuji Itoh, Kenya Honda

  SCIENCE 331 (6015) 337 - 341 0036-8075 2011/01 [Refereed][Not invited]
   

  CD4(+) T regulatory cells (T(regs)), which express the Foxp3 transcription factor, play a critical role in the maintenance of immune homeostasis. Here, we show that in mice, T(regs) were most abundant in the colonic mucosa. The spore-forming component of indigenous intestinal microbiota, particularly clusters IV and XIVa of the genus Clostridium, promoted T(reg) cell accumulation. Colonization of mice by a defined mix of Clostridium strains provided an environment rich in transforming growth factor-beta and affected Foxp3(+) T(reg) number and function in the colon. Oral inoculation of Clostridium during the early life of conventionally reared mice resulted in resistance to colitis and systemic immunoglobulin E responses in adult mice, suggesting a new therapeutic approach to autoimmunity and allergy.
• The Ras-PI3K Signaling Pathway Is Involved in Clathrin-Independent Endocytosis and the Internalization of Influenza Viruses

  Yoichiro Fujioka, Masumi Tsuda, Tomoe Hattori, Junko Sasaki, Takehiko Sasaki, Tadaaki Miyazaki, Yusuke Ohba

  PLOS ONE 6 (1) e16324  1932-6203 2011/01 [Refereed][Not invited]
   

  Background: Influenza virus infection causes highly contagious, severe respiratory disorders and gives rise to thousands of deaths every year; however, the efficacy of currently approved defense strategies, including vaccines and neuraminidase inhibitors, is limited because the virus frequently acquires resistance via antigen drift and reassortment. It is therefore important to establish a novel, effective therapeutic strategy that is effective irrespective of viral subtype. Methodology/Principal Findings: Here, we identify the Ras-phosphoinositide 3-kinase (PI3K) signaling pathway as a host-cell regulatory mechanism for influenza virus entry. The binding of Ras to PI3K is specifically involved in clathrinin-dependent endocytosis, endosomal maturation, and intracellular transport of viruses, which result in decreased infectious efficacy of different subtypes of influenza viruses in cells lacking the Ras-PI3K interaction. Moreover, influenza virus infection indeed triggered Ras activation and subsequent PI3K activation in early endosomes. Conclusions/Significance: Taken together, these results demonstrate that the Ras-PI3K signaling axis acts as a host-oriented mechanism for viral internalization. Given that virus incorporation is a process conserved among virus subtypes and species, this signaling pathway may provide a target for potent, well-tolerated prophylactics and therapeutics against a broad range of viruses.
• ZAPS is a potent stimulator of signaling mediated by the RNA helicase RIG-I during antiviral responses

  Sumio Hayakawa, Souichi Shiratori, Hiroaki Yamato, Takeshi Kameyama, Chihiro Kitatsuji, Fumi Kashigi, Showhey Goto, Shoichiro Kameoka, Daisuke Fujikura, Taisho Yamada, Tatsuaki Mizutani, Mika Kazumata, Maiko Sato, Junji Tanaka, Masahiro Asaka, Yusuke Ohba, Tadaaki Miyazaki, Masahiro Imamura, Akinori Takaoka

  NATURE IMMUNOLOGY 12 (1) 37 - U56 1529-2908 2011/01 [Refereed][Not invited]
   

  The poly(ADP-ribose) polymerases (PARPs) participate in many biological and pathological processes. Here we report that the PARP-13 shorter isoform (ZAPS), rather than the full-length protein (ZAP), was selectively induced by 5'-triphosphate-modified RNA (3pRNA) and functioned as a potent stimulator of interferon responses in human cells mediated by the RNA helicase RIG-I. ZAPS associated with RIG-I to promote the oligomerization and ATPase activity of RIG-I, which led to robust activation of IRF3 and NF-kappa B transcription factors. Disruption of the gene encoding ZAPS resulted in impaired induction of interferon-alpha (IFN-alpha), IFN-beta and other cytokines after viral infection. These results indicate that ZAPS is a key regulator of RIG-I signaling during the innate antiviral immune response, which suggests its possible use as a therapeutic target for viral control.
• Induction of Colonic Regulatory T Cells by Indigenous Clostridium Species

  Atarashi Koji, Tanoue Takeshi, Shima Tatsuichiro, Imaoka Akemi, Kuwahara Tomomi, Momose Yoshika, Cheng Genhong, Yamasaki Sho, Saito Takashi, Ohba Yusuke, Taniguchi Tadatsugu, Takeda Kiyoshi, Hori Shohei, Ivanov Ivaylo I, Umesaki Yoshinori, Itoh Kikuji, Honda Kenya

  Science 1 - 5 0036-8075 2010/12/23 [Not refereed][Not invited]
   

  <p>CD4⁺ T regulatory cells (Tregs), expressing the Foxp3 transcription factor, play a critical role in the maintenance of immune homeostasis. Here, we show that in mice, Tregs were most abundant in the colonic mucosa. The spore-forming component of indigenous intestinal microbiota-particularly clusters IV and XIVa of the genus Clostridium, promoted Treg cell accumulation. Colonization of mice by a defined mix of Clostridium strains provided an environment rich in transforming growth factor-β (TGF-β) and affected Foxp3⁺ Treg number and function in the colon. Oral inoculation of Clostridium during the early life of conventionally reared mice resulted in resistance to colitis and systemic IgE responses in adult mice, suggesting a new therapeutic approach to autoimmunity and allergy.</p>
• Conversion of Helicobacter pylori CagA from senescence inducer to oncogenic driver through polarity-dependent regulation of p21

  Yasuhiro Saito, Naoko Murata-Kamiya, Toshiya Hirayama, Yusuke Ohba, Masanori Hatakeyama

  JOURNAL OF EXPERIMENTAL MEDICINE 207 (10) 2157 - 2174 0022-1007 2010/09 [Refereed][Not invited]
   

  The Helicobacter pylori CagA bacterial oncoprotein plays a critical role in gastric carcinogenesis. Upon delivery into epithelial cells, CagA causes loss of polarity and activates aberrant Erk signaling. We show that CagA-induced Erk activation results in senescence and mitogenesis in nonpolarized and polarized epithelial cells, respectively. In nonpolarized epithelial cells, Erk activation results in oncogenic stress, up-regulation of the p21(Waf1/Cip1) cyclin-dependent kinase inhibitor, and induction of senescence. In polarized epithelial cells, CagA-driven Erk signals prevent p21(Waf1/Cip1) expression by activating a guanine nucleotide exchange factor-H1-RhoA-RhoA-associated kinase-c-Myc pathway. The microRNAs miR-17 and miR-20a, induced by c-Myc, are needed to suppress p21(Waf1/Cip1) expression. CagA also drives an epithelial-mesenchymal transition in polarized epithelial cells. These findings suggest that CagA exploits a polarity-signaling pathway to induce oncogenesis.
• A Novel FRET-Based Biosensor for the Measurement of BCR-ABL Activity and Its Response to Drugs in Living Cells

  Tatsuaki Mizutani, Takeshi Kondo, Stephanie Darmanin, Masumi Tsuda, Shinya Tanaka, Minoru Tobiume, Masahiro Asaka, Yusuke Ohba

  CLINICAL CANCER RESEARCH 16 (15) 3964 - 3975 1078-0432 2010/08 [Refereed][Not invited]
   

  Purpose: To develop a novel diagnostic method for the assessment of drug efficacy in chronic myeloid leukemia (CML) patients individually, we generated a biosensor that enables the evaluation of BCR-ABL kinase activity in living cells using the principle of fluorescence resonance energy transfer (FRET). Experimental Design: To develop FRET-based biosensors, we used CrkL, the most characteristic substrate of BCR-ABL, and designed a protein in which CrkL is sandwiched between Venus, a variant of YFP, and enhanced cyan fluorescent protein, so that CrkL intramolecular binding of the SH2 domain to phosphorylated tyrosine (Y207) increases FRET efficiency. After evaluation of the properties of this biosensor by comparison with established methods including Western blotting and flow cytometry, BCR-ABL activity and its response to drugs were examined in CML patient cells. Results: After optimization, we obtained a biosensor that possesses higher sensitivity than that of established techniques with respect to measuring BCR-ABL activity and its suppression by imatinib. Thanks to its high sensitivity, this biosensor accurately gauges BCR-ABL activity in relatively small cell numbers and can also detect <1% minor drug-resistant populations within heterogeneous ones. We also noticed that this method enabled us to predict future onset of drug resistance as well as to monitor the disease status during imatinib therapy, using patient cells. Conclusion: In consideration of its quick and practical nature, this method is potentially a promising tool for the prediction of both current and future therapeutic responses in individual CML patients, which will be surely beneficial for both patients and clinicians. Clin Cancer Res; 16(15); 3964-75. (C) 2010 AACR.
• 腫瘍血管内皮細胞と腫瘍細胞との相互作用解析(Analysis of interaction between tumor endothelial cells and tumor cells)

  間石 奈湖, 大賀 則孝, 秋山 廣輔, 北山 和子, 近藤 美弥子, 川本 泰輔, 大澤 崇宏, 山本 和幸, 樋田 泰浩, 大場 雄介, 進藤 正信, 樋田 京子

  日本癌学会総会記事 69回 103 - 103 0546-0476 2010/08 [Refereed][Not invited]
  
• Visualization of Ras-PI3K interaction in the endosome using BiFC

  Kaori Tsutsumi, Yoichiro Fujioka, Masumi Tsuda, Hideaki Kawaguchi, Yusuke Ohba

  CELLULAR SIGNALLING 21 (11) 1672 - 1679 0898-6568 2009/11 [Refereed][Not invited]
   

  Recent studies indicate the importance of spatiotemporal regulation in the diversity and specificity of intracellular signaling. Here, we show that Ras-PI3K signaling plays an important role in the local regulation of phosphatidylinositol metabolism in the endosome through live-cell imaging by using a bimolecular fluorescence complementation technique, in which molecular interaction is indicated by fluorescence emission. Using several possible combinations of Ras and the Ras-binding domain, we identified an optimal set of probe molecules that yielded the most significant increase in fluorescence intensity between the active and inactive forms of Ras. This combination revealed that, among the Ras effectors tested, phosphatidy-linositol 3-kinase (PI3K) was specifically implicated in signaling in the endosome. We also found that full length PI3K was recruited to the endosome in EGF- and Ras-dependent manners, which appears to be essential for the activation of PI3K in this compartment. Taken together, these findings demonstrate that the spatiotemporal regulation of Ras-PI3K signaling may dictate the activation of PI3K and subsequent downstream signaling in the endosome. (C) 2009 Elsevier Inc. All rights reserved.
• Basics of bioimaging and its applications

  津田 真寿美, 大場 雄介

  The Cell ニューサイエンス社 41 (11) 462 - 465 1346-7557 2009/10 [Not refereed][Not invited]
  
• Adaptor Protein Crk Induces Src-Dependent Activation of p38 MAPK in Regulation of Synovial Sarcoma Cell Proliferation

  Takuya Watanabe, Masumi Tsuda, Shinya Tanaka, Yusuke Ohba, Hideaki Kawaguchi, Tokifumi Majima, Hirofumi Sawa, Akio Minami

  MOLECULAR CANCER RESEARCH 7 (9) 1582 - 1592 1541-7786 2009/09 [Refereed][Not invited]
   

  The adaptor protein Crk mediates intracellular signaling related to cell motility and proliferation and is implicated in human tumorigenesis. The role of Crk in the growth of human sarcoma has remained unclear, however. The present study shows that Crk-induced activation of Src and subsequent signaling by p38 mitogen-activated protein kinase (MAPK) contribute to the enhanced proliferation of human synovial sarcoma cells. Depletion of Crk by RNA interference markedly inhibited proliferation of the synovial sarcoma cell lines HS-SYII, SYO-1, and Fuji as well as prevented anchorage-independent growth. Conversely, reconstitution with CrkII by authentic small interfering RNA-resistant Crk gene restored proliferation in Crk-silenced SYO-1 cells. Crk-depleted synovial sarcoma cells manifested enhanced transcriptional activity and expression of the p16(INK4A) gene, resulting in their accumulation in G, phase of the cell cycle. In response to hepatocyte growth factor stimulation, Crk prominently induced the tyrosine phosphorylation of Grb2-associated binder 1 through activation of Src and focal adhesion kinase, and the Src family kinase inhibitor PP2 almost completely inhibited the proliferation of SYO-1 cells. Crk also induced the phosphorylation of p38 MAPK, and SB203580, a p38 MAPK-specific inhibitor, increased expression of p16(INK4A) gene in SYO-1 cells. Furthermore, SB203580 or depletion of p38 MAPK by small interfering RNA suppressed both the phosphorylation of Akt triggered by hepatocyte growth factor and the proliferation of SYO-1 cells. These results suggest that Crk promotes proliferation of human synovial sarcoma cells through activation of Src and its downstream signaling by a novel p38 MAPK-Akt pathway, with these signaling molecules providing potent new targets for molecular therapeutics. (Mol Cancer Res 2009;7(9):1582-92)
• Helicobacter pylori CagA Causes Mitotic Impairment and Induces Chromosomal Instability

  Mayumi Umeda, Naoko Murata-Kamiya, Yasuhiro Saito, Yusuke Ohba, Masayuki Takahashi, Masanori Hatakeyama

  JOURNAL OF BIOLOGICAL CHEMISTRY 284 (33) 22166 - 22172 0021-9258 2009/08 [Refereed][Not invited]
   

  Infection with cagA-positive Helicobacter pylori is the strongest risk factor for the development of gastric carcinoma. The cagA gene product CagA, which is delivered into gastric epithelial cells, specifically binds to and aberrantly activates SHP-2 oncoprotein. CagA also interacts with and inhibits partitioning-defective 1 (PAR1)/MARK kinase, which phosphorylates microtubule-associated proteins to destabilize microtubules and thereby causes epithelial polarity defects. In light of the notion that microtubules are not only required for polarity regulation but also essential for the formation of mitotic spindles, we hypothesized that CagA-mediated PAR1 inhibition also influences mitosis. Here, we investigated the effect of CagA on the progression of mitosis. In the presence of CagA, cells displayed a delay in the transition from prophase to metaphase. Furthermore, a fraction of the CagA-expressing cells showed spindle misorientation at the onset of anaphase, followed by chromosomal segregation with abnormal division axis. The effect of CagA on mitosis was abolished by elevated PAR1 expression. Conversely, inhibition of PAR1 kinase elicited mitotic delay similar to that induced by CagA. Thus, CagA-mediated inhibition of PAR1, which perturbs microtubule stability and thereby causes microtubule-based spindle dysfunction, is involved in the prophase/metaphase delay and subsequent spindle misorientation. Consequently, chronic exposure of cells to CagA induces chromosomal instability. Our findings reveal a bifunctional role of CagA as an oncoprotein: CagA elicits uncontrolled cell proliferation by aberrantly activating SHP-2 and at the same time induces chromosomal instability by perturbing the microtubule-based mitotic spindle. The dual function of CagA may cooperatively
• Parkin stabilizes PINK1 through direct interaction

  Kahori Shiba, Takeo Arai, Shigeto Sato, Shin-ichiro Kubo, Yusuke Ohba, Yoshikuni Mizuno, Nobutaka Hattori

  BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 383 (3) 331 - 335 0006-291X 2009/06 [Refereed][Not invited]
   

  Parkinson disease (PD) is the most common movement disorder and is characterized by dopaminergic dysfunction. The majority of PD cases are sporadic: however, the discovery of genes linked to rare familial forms of the disease has provided crucial insight into the molecular mechanisms of disease pathogenesis. Multiple genes mediating familial forms of Parkinson's disease (PD) have been identified, such as parkin (PARK2) and phosphatase and tensin homologue deleted on chromosome ten (PTEN)-induced putative kinase 1: PINK1 (PARK6). Here, we showed that Parkin directly interacts with PINK1, but did not bind to pathogenic PINK1 mutants. Parkin, but not its pathogenic mutants, stabilizes PINK1 by interfering with its degradation via the ubiquitin-mediated proteasomal pathway. In addition, the interaction between Parkin and PINK1 resulted in reciprocal reduction of their solubility. Our results indicate that Parkin regulates PINK1 stabilization via direct interaction with PINK1, and operates through a common pathway with PINK1 in the pathogenesis of early-onset PD. (C) 2009 Elsevier Inc. All rights reserved.
• Transcription factor 8 activates R-Ras to regulate angiogenesis

  Takayuki Inuzuka, Masumi Tsuda, Hideaki Kawaguchi, Yusuke Ohba

  BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 379 (2) 510 - 513 0006-291X 2009/02 [Refereed][Not invited]
   

  We have recently reported that transcription factor 8 (TCF8) negatively regulates pathological angiogenesis by regulating endothelial invasiveness by acting as a transcriptional attenuator of matrix metalloproteinase 1. TCF8 also modulates cell-matrix and cell-cell adhesion; however molecular mechanism of this TCF8 function remains obscure. Here, we provide evidence that TCF8 activates R-Ras, another class of angiogenic regulator, to suppress angiogenesis by a mechanism other than a transcriptional attenuator. Tube formation by human umbilical vein endothelial cells (HUVECs) facilitated by TCF8 suppression was significantly inhibited by the expression of constitutive active mutant of R-Ras. When we examined the mRNA expression levels of R-Ras regulators, no significant changes were observed to explain the R-Ras activation by TCF8. Interestingly, we found that TCF8 bound to CalDAG-GEFIII, an R-Ras activator, in the cytosol, indicating that TCF8 emanates signaling for R-Ras activation from cytosol to regulate angiogenesis negatively. (C) 2008 Elsevier Inc. All rights reserved.
• Integral Role of Transcription Factor 8 in the Negative Regulation of Tumor Angiogenesis

  Takayuki Inuzuka, Masumi Tsuda, Shinya Tanaka, Hideaki Kawaguchi, Yujiro Higashi, Yusuke Ohba

  CANCER RESEARCH 69 (4) 1678 - 1684 0008-5472 2009/02 [Refereed][Not invited]
   

  Angiogenesis is involved in various physiologic and pathological conditions, including tumor growth, and is tightly regulated by the orchestration of proangiogenic and antiangiogenic factors. Inhibition of vascular endothelial growth factor (VEGF), the best-established antiangiogenic treatment in cancer, has shown some effectiveness; however, the identification of novel regulators, whose function is independent of VEGF, is required to achieve better outcomes. Here, we show that transcription factor 8 (TCF8) is up-regulated in endothelial cells during angiogenesis, acting as a negative regulator. Furthermore, TCF8 is specifically expressed in the endothelium of tumor vessels. 1cf8-heterozygous knockout mice are more permissive than wildtype mice to the formation of tumor blood vessels in s.c. implanted melanoma, which seems to contribute to the more aggressive growth and the lung metastases of the tumor in mutant mice. Suppression of TCF8 facilitates angiogenesis in both in vitro and ex vivo models, and displays comprehensive cellular phenotypes, including enhanced cell invasion, impaired cell adhesion, and increased cell monolayer permeability due to, at least partly, MMPI overexpression, attenuation of focal adhesion formation, and insufficient VE-cadherin recruitment, respectively. Taken together, our findings define a novel, integral role for TCF8 in the regulation of pathologic angiogenesis, and propose TCF8 as a target for therapeutic intervention in cancer. [Cancer Res 2009;69(4):1678-84]
• Increased Motility and Invasiveness in Tumor Cells That Survive 10 Gy Irradiation

  Kaori Tsutsumi, Masumi Tsuda, Natsuka Yazawa, Hirotaka Nakamura, Seiichiro Ishihara, Hisashi Haga, Motoaki Yasuda, Rie Yamazaki, Hiroki Shirato, Hideaki Kawaguchi, Takeshi Nishioka, Yusuke Ohba

  CELL STRUCTURE AND FUNCTION 34 (2) 89 - 96 0386-7196 2009 [Refereed][Not invited]
   

  Radiotherapy is an important noninvasive treatment for many types of cancer. However, it has been reported that the proliferative, invasive, and metastatic capacities of tumor cells can be increased in the repopulated tumors that survive radiotherapy. We have previously established a radiation-surviving cell model for the human non-small cell lung cancer cell line H1299 by harvesting relic cells 14 days after irradiation (IR cells). Here, we report that cell invasion, cell migration, and cell adhesion are enhanced in these surviving cancer cells. The mRNA expression levels of matrix metalloproteinases (MMPs), including mmp1, mmp2, and mmp9, were upregulated in IR cells compared with parental cells. A gelatin zymogram, wound healing assay, and invasion assay showed increased MMP activity, cell motility, and invasiveness in IR cells, respectively. Moreover, IR cells adhered more tightly to collagen-coated dishes than parental cells. Consistently, paxillin, phosphorylated FAK, integrin beta 1, and vinculin were strongly localized at focal adhesions in IR cells, as visualized by immunofluorescence. In this report, we identify molecules responsible for the malignant properties of tumor cells that survive irradiation. These molecules may be important therapeutic targets for the control of repopulated tumors after radiotherapy.
• Clinicopathological significance of expression of p-c-Jun, TCF4 and beta-Catenin in colorectal tumors

  Kayoko Takeda, Ichiro Kinoshita, Yasushi Shimizu, Yusuke Ohba, Tomoo Itoh, Yoshihiro Matsuno, Toshiaki Shichinohe, Hirotoshi Dosaka-Akita

  BMC CANCER 8 328  1471-2407 2008/11 [Refereed][Not invited]
   

  Background: A recent study has shown that phosphorylated c-Jun (p-c-Jun) interacts with TCF4 to form a complex that cooperatively enhances their transcriptional activity in the presence of beta-Catenin, and that their interaction is critical for mouse intestinal tumorigenesis. To determine the significance of these three proteins in human colorectal tumors, we analyzed their nuclear expression by immunohistochemistry. Methods: we analyzed their nuclear expression by immunohistochemistry using paraffin-embedded specimens of 68 resected colorectal tumors, which consisted of 19 adenomas, 14 high-grade intraepithelial neoplasia (HGINs) and 35 adenocarcinomas. We also analyzed the expression of MMP7, which has functional AP-I and TCF binding sites in its promoter. Results: Expression of p-c-Jun, TCF4 and beta-Catenin were significantly higher in adenomas than in the adjacent normal epithelia. Expression of p-c-Jun and beta-Catenin in HGINs and adenocarcinomas were also significantly higher than in the adjacent normal epithelia. p-c-Jun expression, but not TCF4 and beta-Catenin, was higher in adenomas and HGINs than in adenocarcinomas, in which p-c-Jun expression was negatively correlated with pT stage progression. Furthermore, significant correlations of expression were observed between p-c-Jun and TCF4 ( r = 0.25, p = 0.04), TCF4 and beta-Catenin ( r = 0.30, p = 0.01), p-c-Jun and MMP7 ( r = 0.26, p = 0.03), and TCF4 and MMP7 ( r = 0.39, p = 0.0008), respectively. Conclusion: These results suggest that nuclear expression of p-c-Jun, TCF4 and beta-Catenin have important roles in human colorectal tumor development and that p-c-Jun may play a pivotal role in the earlier stages of tumor development.
• シグナル伝達研究から創薬開発へ 2.細胞内シグナル伝達の可視化とその創薬への応用

  大場雄介, 津田真寿美

  実験医学 26 (15) 2506 - 2513 0288-5514 2008/09/15 [Not refereed][Not invited]
  
• PTHrP promotes malignancy of human oral cancer cell downstream of the EGFR signaling

  Tamaki Yamada, Masumi Tsuda, Yusuke Ohba, Hideaki Kawaguchi, Yasunori Totsuka, Masanobu Shindoh

  BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 368 (3) 575 - 581 0006-291X 2008/04 [Not refereed][Not invited]
   

  Parathyroid hormone-related protein (PTHrP) is detected in many aggressive tumors and involved in malignant conversion; however, the underlying mechanism remains obscure. Here, we identified PTHrP as a mediator of epidermal growth factor receptor (EGFR) signaling to promote the malignancies of oral cancers. PTHrP mRNA was abundantly expressed in most of the quiescent oral cancer cells, and was significantly upregulated by EGF stimulation via ERK and p38 MAPK. PTHrP silencing by RNA interference, as well as EGFR inhibitor AG1478 treatment, significantly suppressed cell proliferation, migration, and invasiveness. Furthermore, combined treatment of AG1478 and PTHrP knockdown achieved synergistic inhibition of malignant phenotypes. Recombinant PTHrP substantially promoted cell motility, and rescued the inhibition by PTHrP knockdown, suggesting the paracrine/autocrine function of PTHrP. These data indicate that PTHrP contributes to the malignancy of oral cancers downstream of EGFR signaling, and may thus provide a therapeutic target for oral cancer. (c) 2008 Elsevier Inc. All rights reserved.
• PTHrP promotes malignancy of human oral cancer cell downstream of the EGFR signaling

  Tamaki Yamada, Masumi Tsuda, Yusuke Ohba, Hideaki Kawaguchi, Yasunori Totsuka, Masanobu Shindoh

  BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 368 (3) 575 - 581 0006-291X 2008/04 [Refereed][Not invited]
   

  Parathyroid hormone-related protein (PTHrP) is detected in many aggressive tumors and involved in malignant conversion; however, the underlying mechanism remains obscure. Here, we identified PTHrP as a mediator of epidermal growth factor receptor (EGFR) signaling to promote the malignancies of oral cancers. PTHrP mRNA was abundantly expressed in most of the quiescent oral cancer cells, and was significantly upregulated by EGF stimulation via ERK and p38 MAPK. PTHrP silencing by RNA interference, as well as EGFR inhibitor AG1478 treatment, significantly suppressed cell proliferation, migration, and invasiveness. Furthermore, combined treatment of AG1478 and PTHrP knockdown achieved synergistic inhibition of malignant phenotypes. Recombinant PTHrP substantially promoted cell motility, and rescued the inhibition by PTHrP knockdown, suggesting the paracrine/autocrine function of PTHrP. These data indicate that PTHrP contributes to the malignancy of oral cancers downstream of EGFR signaling, and may thus provide a therapeutic target for oral cancer. (c) 2008 Elsevier Inc. All rights reserved.
• Weekly paclitaxel/5-fluorouracil followed by platinum retreatment for patients with recurrent ovarian cancer: a single institution experience

  H. Watari, M. Hosaka, T. Mitamura, M. Moriwaki, Y. Ohba, Y. Todo, M. Takeda, Y. Ebina, N. Sakuragi

  EUROPEAN JOURNAL OF GYNAECOLOGICAL ONCOLOGY 29 (6) 573 - 577 0392-2936 2008 [Refereed][Not invited]
   

  Purpose: Since the prognosis of recurrent ovarian cancer patients is still poor, we need to establish a useful treatment strategy to achieve their long-term survival. We treated recurrent ovarian cancer patients with weekly paclitaxel (PTX)/5-fluorouracil (5-FU) followed by platinum retreatment to investigate its clinical efficacy in a preliminary manner. Methods: Sixteen patients with recurrent ovarian cancer. pretreated with taxane and platinum, were treated with weekly paclitaxel (PTX)/5-fluorouracil (FU). PTX (80 mg/m(2)) on day 1, 8, and 15 was combined with a bolus injection of 5-FU (500 mg/m(2)) on day 2, 9, and 16. Chemotherapy was given every four weeks. Patients with stable disease or progressive disease Were subsequently retreated with a platinum-containing regimen, Response was evaluated by RECIST criteria or CA 1225 criteria. Toxicities were evaluated according to the National Cancer Institute-common toxicity Criteria (NCI-CTC) version 3. Results: Among live patients with sensitive disease, one Of four patients with measurable turner and one without measurable tumor responded to weekly PTX/5-FU. Among 11 patients with resistant disease. none of five patients with measurable tumor and three of six patients without measurable tumor responded to weekly PTX/5-FU, Overall objective response rate by weekly PTX/5-FU was 31.3% (5/16). Among 16 patients, 13 patients who showed no response or progressive disease (three with sensitive disease, ten with resistant disease) received platinum retreatment after weekly PTX/5FU. All three patients with sensitive disease and three of ten patients with resistant disease revealed response to platinum retreatment. Overall objective response rate by platinum retreatment after weekly PTX/5-FU was 46.2% (6/13). Conclusions: Weekly PTX/5FU followed by platinum retreatment Could be a useful treatment strategy for recurrent ovarian cancer patients. We need to establish the standard treatment strategy for recurrent ovarian cancer patients with a poor prognosis.
• Homeostatic erythropoiesis by the transcription factor IRF2 through attenuation of type I interferon signaling

  Tatsuaki Mizutani, Kohichiro Tsuji, Yasuhiro Ebihara, Shinsuke Taki, Yusuke Ohba, Tadatsugu Taniguchi, Kenya Honda

  EXPERIMENTAL HEMATOLOGY 36 (3) 255 - 264 0301-472X 2008 [Refereed][Not invited]
   

  Objective. Erythrocyte production is tightly regulated by cytokines, particularly erythropoietin (EPO), which affects expansion and viability of erythroid lineage cells via induction of several factors, including Bcl2-like 1 (Bcl-XL). Because type I interferon (IFN) is known to inhibit erythropoiesis, we studied mice deficient in the gene for interferon regulatory factor 2 (IRF2), which functions as a negative regulator of type I IFN signaling, in the context of erythropoiesis regulation. Materials and Methods. We performed hematologic analyses and detected normocytic anemia in Irj2-deficient mice. Results. Assessment of the maturation of erythroid progenitors in Irf2-deficient bone marrow by flow cytometry revealed a decreased number of late erythroblasts accompanied by an increased number of early erythroid progenitors. Irf2-deficient mice manifested elevated serum EPO levels, decreased Bcl-XL expression levels and enhanced apoptosis of erythroblasts, which may account for the decreased number of late erythroblasts. We further assessed the role of IRF2 in the regulation of type I IFN signaling during erythropoiesis, and found that additional homozygous mutation of IFNAR1, a subunit of type I IFN receptor complex, led to rescue of the defect of erythropoiesis in Irf2-deficient mice. Conclusions. Impaired erythropoiesis in Irf2-deficient mice results from excessive type I IFN signaling, which inhibits Bcl-XL expression in erythroid lineage cells. Our present study provides a mechanistic understanding of the potential cross-talk between type I IFN and EPO signaling pathways during erythropoiesis and may offer therapeutic insights into anemia. (c) 2008 ISEH - Society for Hematology and Stem Cells. Published by Elsevier Inc.
• Spatiotemporal mobilization of Toll/IL-1 receptor domain-containing adaptor molecule-1 in response to dsRNA

  Kenji Funami, Miwa Sasai, Yusuke Ohba, Hiroyuki Oshiumi, Tsukasa Seya, Misako Matsumoto

  JOURNAL OF IMMUNOLOGY 179 (10) 6867 - 6872 0022-1767 2007/11 [Refereed][Not invited]
   

  TLR3 recognizes viral dsRNA and induces antiviral immune responses. TLR3-mediated cell activation relies on Toll/IL-1R (TIR) domain-containing adaptor molecule-1 (TICAM-1, also named TIR domain-containing adaptor inducing IFN-beta or TRIF), which recruits downstream signaling molecules to activate the transcription factors IFN regulatory factor 3 (IRF-3) and NF-kappa B. The mechanisms by which TICAM-1 is activated and transmits signals remain largely unknown. In this study we show that TICAM-1 alters its distribution profile from a diffuse cytoplasmic form to a speckle-like structure in response to dsRNA. The receptor-interacting protein 1 (RIP1), a crucial signaling molecule for TICAM-1-mediated NF-kappa B activation, accumulated in the TICAM-1 speckles. In addition, NF-kappa B-activating kinase-associated protein 1 (NAP1), a downstream molecule linking TICAM-1 and the IRF-3-activating kinase TBK1 (TANK-binding kinase 1), was also recruited to the TICAM-1 speckles. Notably, a transient colocalization of TICAM-1 and TLR3 was observed before the extensive formation of the TICAM-1 speckles. Thus, the spatiotemporal mobilization of TICAM-1 in response to dsRNA and the formation of the TICAM-1 speckles containing RIP1 and NAP1 are important for the activation of the TLR3-TICAM-1 pathway.
• Oligodendrocyte lineage transcription factor 2 inhibits the motility of a human glial tumor cell line by activating RhoA

  Kouichi Tabu, Yusuke Ohba, Tadaki Suzuki, Yoshinori Makino, Taichi Kimura, Akiko Ohnishi, Mieko Sakai, Takuya Watanabe, Shinya Tanaka, Hirofurni Sawa

  MOLECULAR CANCER RESEARCH 5 (10) 1099 - 1109 1541-7786 2007/10 [Not refereed][Not invited]
   

  The basic helix-loop-helix transcription factor, oligodendrocyte lineage transcription factor 2 (OLIG2), is specifically expressed in the developing and mature central nervous system and plays an important role in oligodendrogenesis from neural progenitors. It is also expressed in various types of glial tumors, but rarely in glioblastoma. Although we previously showed that OLIG2 expression inhibits glioma cell growth, its role in tumorigenesis remains incompletely understood. Here, we investigated the effect of OLIG2 expression on the migration of the human glioblastoma cell line U12-1. In these cells, OLIG2 expression is controlled by the Tet-off system. Induction of OLIG2 expression inhibited both the migration and invasiveness of U12-1 cells. OLIG2 expression also increased the activity of the GTPase RhoA as well as inducing the cells to form stress fibers and focal adhesions. Experiments using short interfering RNA against p27(KiP1) revealed that up-regulation of the p27(Kip1) protein was not essential for RhoA activation, rather it contributed independently to the decreased motility of OLIG2-expressing U12-1 cells. Alternatively, semiquantitative reverse transcription-PCR analysis revealed that mRNA expression of RhoGAP8, which regulates cell migration, was decreased by OLIG2 expression. Furthermore, expression of C3 transferase, which inhibits Rho via ADP ribosylation, attenuated the OLIG2-induced inhibition of cell motility. Imaging by fluorescence resonance energy transfer revealed that in U12-1 cells lacking OLIG2, the active form of RhoA was localized to protrusions of the cell membrane. In contrast, in OLIG2-expressing cells, it lined almost the entire plasma membrane. Thus, OLIG2 suppresses the motile phenotype of glioblastoma cells by activating RhoA.
• Oligodendrocyte lineage transcription factor 2 inhibits the motility of a human glial tumor cell line by activating RhoA

  Kouichi Tabu, Yusuke Ohba, Tadaki Suzuki, Yoshinori Makino, Taichi Kimura, Akiko Ohnishi, Mieko Sakai, Takuya Watanabe, Shinya Tanaka, Hirofurni Sawa

  MOLECULAR CANCER RESEARCH 5 (10) 1099 - 1109 1541-7786 2007/10 [Refereed][Not invited]
   

  The basic helix-loop-helix transcription factor, oligodendrocyte lineage transcription factor 2 (OLIG2), is specifically expressed in the developing and mature central nervous system and plays an important role in oligodendrogenesis from neural progenitors. It is also expressed in various types of glial tumors, but rarely in glioblastoma. Although we previously showed that OLIG2 expression inhibits glioma cell growth, its role in tumorigenesis remains incompletely understood. Here, we investigated the effect of OLIG2 expression on the migration of the human glioblastoma cell line U12-1. In these cells, OLIG2 expression is controlled by the Tet-off system. Induction of OLIG2 expression inhibited both the migration and invasiveness of U12-1 cells. OLIG2 expression also increased the activity of the GTPase RhoA as well as inducing the cells to form stress fibers and focal adhesions. Experiments using short interfering RNA against p27(KiP1) revealed that up-regulation of the p27(Kip1) protein was not essential for RhoA activation, rather it contributed independently to the decreased motility of OLIG2-expressing U12-1 cells. Alternatively, semiquantitative reverse transcription-PCR analysis revealed that mRNA expression of RhoGAP8, which regulates cell migration, was decreased by OLIG2 expression. Furthermore, expression of C3 transferase, which inhibits Rho via ADP ribosylation, attenuated the OLIG2-induced inhibition of cell motility. Imaging by fluorescence resonance energy transfer revealed that in U12-1 cells lacking OLIG2, the active form of RhoA was localized to protrusions of the cell membrane. In contrast, in OLIG2-expressing cells, it lined almost the entire plasma membrane. Thus, OLIG2 suppresses the motile phenotype of glioblastoma cells by activating RhoA.
• Location of a possible miRNA processing site in SmD3/SmB nuclear bodies in arabidopsis

  Yoichiro Fujioka, Maki Utsumi, Yusuke Ohba, Yuichiro Watanabe

  PLANT AND CELL PHYSIOLOGY 48 (9) 1243 - 1253 0032-0781 2007/09 [Not refereed][Not invited]
   

  There has been much recent research on the contribution of microRNA (miRNA) in plant organogenesis and hormone action. In plants, it has been reported that Dicer-like 1 (DCL1), HYPONASTIC LEAVES1 (HYL1). and SERRATE (SE) are involved in the production of miRNAs. The means by which miRNAs are processed and transported is. not well understood in detail, however. In this study, we investigated the intracellular localization and intermolecular interaction of these molecules using imaging techniques, including bimolecular fluorescence complementation and fluorescence resonance energy transfer techniques, making use of various enhanced fluorescent proteins. We found that DCL1, HYL1 and SE formed bodies which localized in the nuclei. We were also able to locate the miRNA primary transcript using an MS2-tagged method on these bodies. It appears very likely that the observed DCL1-HYL1-SE nuclear body is involved in miRNA production. Co-expression of SmD3 or SmB proteins revealed the localization of DCL1-HYL1-SE complexes in the SmD3/SmB nuclear bodies.
• Location of a possible miRNA processing site in SmD3/SmB nuclear bodies in arabidopsis

  Yoichiro Fujioka, Maki Utsumi, Yusuke Ohba, Yuichiro Watanabe

  PLANT AND CELL PHYSIOLOGY 48 (9) 1243 - 1253 0032-0781 2007/09 [Refereed][Not invited]
   

  There has been much recent research on the contribution of microRNA (miRNA) in plant organogenesis and hormone action. In plants, it has been reported that Dicer-like 1 (DCL1), HYPONASTIC LEAVES1 (HYL1). and SERRATE (SE) are involved in the production of miRNAs. The means by which miRNAs are processed and transported is. not well understood in detail, however. In this study, we investigated the intracellular localization and intermolecular interaction of these molecules using imaging techniques, including bimolecular fluorescence complementation and fluorescence resonance energy transfer techniques, making use of various enhanced fluorescent proteins. We found that DCL1, HYL1 and SE formed bodies which localized in the nuclei. We were also able to locate the miRNA primary transcript using an MS2-tagged method on these bodies. It appears very likely that the observed DCL1-HYL1-SE nuclear body is involved in miRNA production. Co-expression of SmD3 or SmB proteins revealed the localization of DCL1-HYL1-SE complexes in the SmD3/SmB nuclear bodies.
• DAI (DLM-1/ZBP1) is a cytosolic DNA sensor and an activator of innate immune response

  Akinori Takaoka, ZhiChao Wang, Myoung Kwon Choi, Hideyuki Yanai, Hideo Negishi, Tatsuma Ban, Yan Lu, Makoto Miyagishi, Tatsuhiko Kodama, Kenya Honda, Yusuke Ohba, Tadatsugu Taniguchi

  NATURE 448 (7152) 501 - U14 0028-0836 2007/07 [Not refereed][Not invited]
   

  Central to innate immunity is the sensing of pathogen-associated molecular patterns by cytosolic and membrane-associated receptors(1-4). In particular, DNA is a potent activator of immune responses during infection or tissue damage(5-7), and evidence indicates that, in addition to the membrane-associated Toll-like receptor 9, an unidentified cytosolic DNA sensor(s) can activate type I interferon (IFN) and other immune responses(8-10). Here we report on a candidate DNA sensor, previously named DLM-1 ( also called Z-DNA binding protein 1 (ZBP1))(11), for which biological function had remained unknown; we now propose the alternative name DAI (DNA-dependent activator of IFN-regulatory factors(12)). The artificial expression of otherwise IFN-inducible DAI (DLM-1/ ZBP1) in mouse fibroblasts selectively enhances the DNA-mediated induction of type I IFN and other genes involved in innate immunity. On the other hand, RNA interference of messenger RNA for DAI (DLM-1/ ZBP1) in cells inhibits this gene induction programme upon stimulation by DNA from various sources. Moreover, DAI (DLM-1/ZBP1) binds to double-stranded DNA and, by doing so, enhances its association with the IRF3 transcription factor and the TBK1 serine/threonine kinase. These observations underscore an integral role of DAI (DLM-1/ZBP1) in the DNA-mediated activation of innate immune responses, and may offer new insight into the signalling mechanisms underlying DNA- associated antimicrobial immunity and autoimmune disorders.
• Oleamide derivatives suppress the spontaneous metastasis by inhibiting connexin 26

  Yusuke Ohba, Yukiko Kanao, Nobuyoshi Morita, Eri Fujii, Mai Hohrai, Mayuko Takatsuji, Hideki Hirose, Daisaku Miura, Akihiro Watari, Masuo Yutsudo, Hanjun Zhao, Norikazu Yabuta, Akihiko Ito, Yasuyuki Kita, Hiroshi Nojima

  International Journal of Cancer 121 (1) 47 - 54 0020-7136 2007/07/01 [Refereed][Not invited]
   

  We previously reported that overexpressing connexin 26 (Cx26) enhances the spontaneous metastasis of mouse BL6 melanoma cells. In contrast, daily intraperitoneal injections of an oleamide derivative named MI-18 potently inhibits the spontaneous metastasis of BL6 cells. In the present study, we chemically synthesized a novel oleamide derivative named MI-22 and found that it also efficiently suppressed the spontaneous metastasis of BL6 cells. Both MI-18 and MI-22 inhibited the gap junction-mediated intercellular communications (GJIC) that are formed between HeLa cells by the ectopic expression of the hCx26 and hCx32 human connexin subtypes however, they had no effect on GJIC mediated by hCx40, hCx43 or hCx45. Fluorescently labeled MI-18 primarily localized not only at plasma membrane but also at Golgi/endosome. This suggests that this oleamide derivative may also act on the Cx26 molecules that accumulate in the Golgi/endosome because of their overexpression. Notably, neither derivative had a cytotoxic effect on HeLa cells when they were added into the tissue culture medium. Taken together, we propose that the MI-18 and MI-22 oleamide derivatives may serve as prototypes for novel and clinically important anticancer drugs. © 2007 Wiley-Liss, Inc.
• Synthesis of N-functionalized oleamide derivatives

  Yusuke Ohba, Yukiko Kanao, Mayuko Takatsuji, Motoki Ito, Norikazu Yabuta, Hiroshi Nojima, Yasuyuki Kita

  Tetrahedron 63 (18) 3754 - 3761 0040-4020 2007/04/30 [Refereed][Not invited]
   

  Oleamide is an interesting compound, which shows various pharmacological activities including the inhibitory effect of gap junction formation. Recently, the studies of the gap junction have been some of the hot topics in biology and its inhibitors have become more useful tools [Cravatt, B. F. Garcia, O. P. Siuzdak, G. Gilula, N. B. Henriksen, S. J. Boger, D. L. Lerner, R. A. Science 1995, 268, 1506-1509 Cravatt, B. F. Lerner, R. A. Boger, D. L. J. Am. Chem. Soc. 1996, 118, 580-590 Guan, X Cravatt, B. F. Ehring, G. R. Hall, J. E. Boger, D. L. Lerner, R. A. Gilula, N. B. J. Cell Biol. 1997, 139, 1785-1792 Boger, D. L. Patterson, J. E. Guan, X. Cravatt, B. F. Lerner, R. A. Gilula, N. B. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 4810-4815 Ito, A. Morita, N. Miura, D. Koma, Y. Kataoka, T. R. Yamasaki, H. Kitamura, Y. Kita, Y. Nojima, H. Carcinogenesis 2004, 25, 2015-2022]. However, many reports suggest that the functionalizations of oleamide to retain its biological activity were difficult [Boger, D. L. Patterson, J. E. Guan, X. Cravatt, B. F. Lerner, R. A. Gilula, N. B. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 4810-4815 Ito, A. Morita, N. Miura, D. Koma, Y. Kataoka, T. R. Yamasaki, H. Kitamura, Y. Kita, Y. Nojima, H. Carcinogenesis 2004, 25, 2015-2022]. The synthesis of the functionalized oleamide derivatives, whose biological activity is not blocked, has become attractive in both the biological and chemical fields. Herein, by linking the fluorophore to the oleamide by alkyl chains, we synthesized the fluorescently tagged oleamide whose biological feature is similar to that of oleamide. Moreover, we also synthesized other bioactive derivatives tagged by other groups such as the sugars and biotin via alkyl chain linkers. © 2007 Elsevier Ltd. All rights reserved.
• Differential ras activation between caveolae/raft and non-raft Microdomains

  Takashi Fukano, Asako Sawano, Yusuke Ohba, Michiyuki Matsuda, Atsushi Miyawaki

  CELL STRUCTURE AND FUNCTION 32 (1) 9 - 15 0386-7196 2007 [Not refereed][Not invited]
   

  Although the consequences of Ras activation have been studied extensively in the context of oncogenesis, its regulation in physiological modes of signal transduction is not well understood. A fluorescent indicator, Raichu- Ras, was fused to the C- terminal hypervariable regions of H- Ras and K- Ras to create indicators for Ras activation within caveolae/ rafts ( Raichu- tH) and non- raft domains ( Raichu- tK) of the plasma membrane, respectively. Raichu- tH was also found abundantly in endomembranes. To monitor Ras activation with high spatial resolution, it is imperative to observe sectioned images of the signals. We have developed a wide-field fluorescence microscope equipped with a digital micromirror device ( DMD) to acquire optically sectioned images using fringe projection. This system provides reliable signals from fluorescence resonance energy transfer ( FRET) between cyan and yellow mutants of green fluorescent protein. We have used this system to demonstrate that, upon stimulation with growth factors, the two indicators are activated in spatially and temporally unique patterns.
• Spatiotemporal Mobilization of Toll/IL-1 Receptor Domain-Containing Adaptor Molecule-1 in Response to dsRNA.

  J. Immunol. 179 (10) 6867 - 6872 2007 [Not refereed][Not invited]
  
• Differential ras activation between caveolae/raft and non-raft Microdomains

  Takashi Fukano, Asako Sawano, Yusuke Ohba, Michiyuki Matsuda, Atsushi Miyawaki

  CELL STRUCTURE AND FUNCTION 32 (1) 9 - 15 0386-7196 2007 [Refereed][Not invited]
   

  Although the consequences of Ras activation have been studied extensively in the context of oncogenesis, its regulation in physiological modes of signal transduction is not well understood. A fluorescent indicator, Raichu- Ras, was fused to the C- terminal hypervariable regions of H- Ras and K- Ras to create indicators for Ras activation within caveolae/ rafts ( Raichu- tH) and non- raft domains ( Raichu- tK) of the plasma membrane, respectively. Raichu- tH was also found abundantly in endomembranes. To monitor Ras activation with high spatial resolution, it is imperative to observe sectioned images of the signals. We have developed a wide-field fluorescence microscope equipped with a digital micromirror device ( DMD) to acquire optically sectioned images using fringe projection. This system provides reliable signals from fluorescence resonance energy transfer ( FRET) between cyan and yellow mutants of green fluorescent protein. We have used this system to demonstrate that, upon stimulation with growth factors, the two indicators are activated in spatially and temporally unique patterns.
• ウイルス感染と宿主応答・宿主側の要因 MyD88依存的TLR下流シグナル経路におけるIRF-1の役割

  根岸 英雄, 柳井 秀元, 坂口 信也, 藤田 靖之, 篠原 正浩, 高柳 広, 大場 雄介, 谷口 維紹, 本田 賢也

  日本免疫学会総会・学術集会記録 36 207 - 207 0919-1984 2006/11
• Evidence for licensing of IFN-gamma-induced IFN regulatory factor 1 transcription factor by MyD88 in Toll-like receptor-dependent gene induction program

  Hideo Negishi, Yasuyuki Fujita, Hideyuki Yanai, Shinya Sakaguchi, Xinshou Ouyang, Masahiro Shinohara, Hiroshi Takayanagi, Yusuke Ohba, Tadatsugu Taniguchi, Kenya Honda

  PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 103 (41) 15136 - 15141 0027-8424 2006/10 [Not refereed][Not invited]
   

  The recognition of microbial components by Toll-like receptors (TLRs) initiates signal transduction pathways, which trigger the expression of a series of target genes. It has been reported that TLR signaling is enhanced by cytokines such as IFN-gamma, but the mechanisms underlying this enhancement remain unclear. The MyD88 adaptor, which is essential for signaling by many TILRs, recruits members of the IFN regulatory factor (IRF) family of transcription factors, such as IRF5 and IRF7, to evoke the activation of TLR target genes. In this study we demonstrate that IRF1, which is induced by IFN-gamma, also interacts with and is activated by MyD88 upon TLR activation. We provide evidence that MyD88-associated IRF1 migrates into the nucleus more efficiently than non-MyD88-associated IRF1 and that this IRF1 selectively participates in the TLR-dependent gene induction program. The critical role of MyID88-clepenclent "IRF1 licensing" is underscored by the observation that the induction of a specific gene subset downstream of the TLR-MyD88 pathway, such as IFN-beta, inducible NO synthase, and IL-12p35, are impaired in Irf1-deficient cells. Thus, our present study places IRF1 as an additional member participating in MyD88 signaling and provides a mechanistic insight into the enhancement of the TILR-dependent gene induction program by IFN-gamma.
• Differential contribution of Puma and Noxa in dual regulation of p53-mediated apoptotic pathways

  Tsukasa Shibue, Saori Suzuki, Hideaki Okamoto, Hiroki Yoshida, Yusuke Ohba, Akinori Takaoka, Tadatsugu Taniguchi

  EMBO JOURNAL 25 (20) 4952 - 4962 0261-4189 2006/10 [Not refereed][Not invited]
   

  The activation of tumor suppressor p53 induces apoptosis or cell cycle arrest depending on the state and type of cell, but it is not fully understood how these different responses are regulated. Here, we show that Puma and Noxa, the well- known p53-inducible proapoptotic members of the Bcl-2 family, differentially participate in dual pathways of the induction of apoptosis. In normal cells, Puma but not Noxa induces mitochondrial outer membrane permeabilization (MOMP), and this function is mediated in part by a pathway that involves calcium release from the endoplasmic reticulum (ER) and the subsequent caspase activation. However, upon E1A oncoprotein expression, cells also become susceptible to MOMP induction by Noxa, owing to their sensitization to the ER-independent pathway. These findings offer a new insight into differential cellular responses induced by p53, and may have therapeutic implications in cancer.
• Evidence for licensing of IFN-gamma-induced IFN regulatory factor 1 transcription factor by MyD88 in Toll-like receptor-dependent gene induction program

  Hideo Negishi, Yasuyuki Fujita, Hideyuki Yanai, Shinya Sakaguchi, Xinshou Ouyang, Masahiro Shinohara, Hiroshi Takayanagi, Yusuke Ohba, Tadatsugu Taniguchi, Kenya Honda

  PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 103 (41) 15136 - 15141 0027-8424 2006/10 [Refereed][Not invited]
   

  The recognition of microbial components by Toll-like receptors (TLRs) initiates signal transduction pathways, which trigger the expression of a series of target genes. It has been reported that TLR signaling is enhanced by cytokines such as IFN-gamma, but the mechanisms underlying this enhancement remain unclear. The MyD88 adaptor, which is essential for signaling by many TILRs, recruits members of the IFN regulatory factor (IRF) family of transcription factors, such as IRF5 and IRF7, to evoke the activation of TLR target genes. In this study we demonstrate that IRF1, which is induced by IFN-gamma, also interacts with and is activated by MyD88 upon TLR activation. We provide evidence that MyD88-associated IRF1 migrates into the nucleus more efficiently than non-MyD88-associated IRF1 and that this IRF1 selectively participates in the TLR-dependent gene induction program. The critical role of MyID88-clepenclent "IRF1 licensing" is underscored by the observation that the induction of a specific gene subset downstream of the TLR-MyD88 pathway, such as IFN-beta, inducible NO synthase, and IL-12p35, are impaired in Irf1-deficient cells. Thus, our present study places IRF1 as an additional member participating in MyD88 signaling and provides a mechanistic insight into the enhancement of the TILR-dependent gene induction program by IFN-gamma.
• Differential contribution of Puma and Noxa in dual regulation of p53-mediated apoptotic pathways

  Tsukasa Shibue, Saori Suzuki, Hideaki Okamoto, Hiroki Yoshida, Yusuke Ohba, Akinori Takaoka, Tadatsugu Taniguchi

  EMBO JOURNAL 25 (20) 4952 - 4962 0261-4189 2006/10 [Refereed][Not invited]
   

  The activation of tumor suppressor p53 induces apoptosis or cell cycle arrest depending on the state and type of cell, but it is not fully understood how these different responses are regulated. Here, we show that Puma and Noxa, the well- known p53-inducible proapoptotic members of the Bcl-2 family, differentially participate in dual pathways of the induction of apoptosis. In normal cells, Puma but not Noxa induces mitochondrial outer membrane permeabilization (MOMP), and this function is mediated in part by a pathway that involves calcium release from the endoplasmic reticulum (ER) and the subsequent caspase activation. However, upon E1A oncoprotein expression, cells also become susceptible to MOMP induction by Noxa, owing to their sensitization to the ER-independent pathway. These findings offer a new insight into differential cellular responses induced by p53, and may have therapeutic implications in cancer.
• Negative regulation of Toll-like-receptor signaling by IRF-4

  H Negishi, Y Ohba, H Yanai, A Takaoka, K Honma, K Yui, T Matsuyama, T Taniguchi, K Honda

  PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 102 (44) 15989 - 15994 0027-8424 2005/11 [Refereed][Not invited]
   

  The recognition of microbial components by Toll-like receptors (TLRs) is an event central to the activation of innate and adaptive immune systems. TLR activation triggers the induction of downstream target genes, wherein the TLR-interacting adaptor molecule MyD88 recruits various signaling molecules and transcription factors. Two members of the IFN regulatory factor (IRF) family of transcription factors, IRF-5 and IRF-7, interact with MyD88 and induce proinflammatory cytokines and type I IFNs, respectively. Here, we show that IRF-4 also interacts with MyD88 and acts as a negative regulator of TLR signaling. IRF-4 mRNA is induced by TLR activation, and IRF-4 competes with IRF-5, but not with IRF-7, for MyD88 interaction. The TLR-dependent induction of proinflammatory cytokines is markedly enhanced in peritoneal macrophages from mice deficient in the Irf4 gene, whereas the induction is inhibited by the ectopic expression of IRF-4 in a macrophage cell line. The critical function of IRF-4 in TLR signaling in vivo is underscored by the observation that Irf4-deficient mice show hypersensitivity to DNA-induced shock, with elevated serum proinflammatory cytokine levels. This study may provide an insight into the complex regulatory mechanisms of MyD88 signaling by IRFs.
• IRF-7 is the master regulator of type-I interferon-dependent immune responses

  K Honda, H Yanai, H Negishi, M Asagiri, M Sato, T Mizutani, N Shimada, Y Ohba, A Takaoka, N Yoshida, T Taniguchi

  NATURE 434 (7034) 772 - 777 0028-0836 2005/04 [Not refereed][Not invited]
   

  The type-I interferon (IFN-alpha/beta) response is critical to immunity against viruses and can be triggered in many cell types by cytosolic detection of viral infection, or in differentiated plasmacytoid dendritic cells by the Toll-like receptor 9 (TLR9) subfamily, which generates signals via the adaptor MyD88 to elicit robust IFN induction(1-4). Using mice deficient in the Irf7 gene (Irf7(-/-) mice), we show that the transcription factor IRF-7 is essential for the induction of IFN-alpha/beta genes via the virus-activated, MyD88-independent pathway and the TLR-activated, MyD88-dependent pathway. Viral induction of MyD88-independent IFN-alpha/beta genes is severely impaired in Irf7(-/-) fibroblasts. Consistently, Irf7(-/-) mice are more vulnerable than Myd88(-/-) mice to viral infection, and this correlates with a marked decrease in serum IFN levels, indicating the importance of the IRF-7-dependent induction of systemic IFN responses for innate antiviral immunity. Furthermore, robust induction of IFN production by activation of the TLR9 subfamily in plasmacytoid dendritic cells is entirely dependent on IRF-7, and this MyD88-IRF-7 pathway governs the induction of CD8(+) T-cell responses. Thus, all elements of IFN responses, whether the systemic production of IFN in innate immunity or the local action of IFN from plasmacytoid dendritic cells in adaptive immunity, are under the control of IRF-7.
• Spatiotemporal regulation of MyD88-IRF-7 signalling for robust type-I interferon induction

  K Honda, Y Ohba, H Yanai, H Negishi, T Mizutani, A Takaoka, C Taya, T Taniguchi

  NATURE 434 (7036) 1035 - 1040 0028-0836 2005/04 [Not refereed][Not invited]
   

  Robust type-I interferon (IFN-alpha/beta) induction in plasmacytoid dendritic cells, through the activation of Toll-like receptor 9 (TLR9), constitutes a critical aspect of immunity(1-6). It is absolutely dependent on the transcription factor IRF-7, which interacts with and is activated by the adaptor MyD88 (ref. 7). How plasmacytoid dendritic cells, but not other cell types (such as conventional dendritic cells), are able to activate the MyD88 IRF-7-dependent IFN induction pathway remains unknown. Here we show that the spatiotemporal regulation of MyD88 IRF-7 signalling is critical for a high-level IFN induction in response to TLR9 activation. The IFN-inducing TLR9 ligand, A/D-type CpG oligodeoxynucleotide (CpG-A)(3,4,8-11), is retained for long periods in the endosomal vesicles of plasmacytoid dendritic cells, together with the MyD88-IRF-7 complex. However, in conventional dendritic cells, CpG-A is rapidly transferred to lysosomal vesicles. We further show that conventional dendritic cells can also mount a robust IFN induction if CpG-A is manipulated for endosomal retention using a cationic lipid. This strategy also allows us to demonstrate endosomal activation of the IFN pathway by the otherwise inactive TLR9 ligand B/K-type oligodeoxynucleotide (CpG-B)(3,4,8-12). Thus, our study offers insights into the regulation of TLR9 signalling in space, potentially suggesting a new avenue for therapeutic intervention.
• IRF-7 is the master regulator of type-I interferon-dependent immune responses

  K Honda, H Yanai, H Negishi, M Asagiri, M Sato, T Mizutani, N Shimada, Y Ohba, A Takaoka, N Yoshida, T Taniguchi

  NATURE 434 (7034) 772 - 777 0028-0836 2005/04 [Refereed][Not invited]
   

  The type-I interferon (IFN-alpha/beta) response is critical to immunity against viruses and can be triggered in many cell types by cytosolic detection of viral infection, or in differentiated plasmacytoid dendritic cells by the Toll-like receptor 9 (TLR9) subfamily, which generates signals via the adaptor MyD88 to elicit robust IFN induction(1-4). Using mice deficient in the Irf7 gene (Irf7(-/-) mice), we show that the transcription factor IRF-7 is essential for the induction of IFN-alpha/beta genes via the virus-activated, MyD88-independent pathway and the TLR-activated, MyD88-dependent pathway. Viral induction of MyD88-independent IFN-alpha/beta genes is severely impaired in Irf7(-/-) fibroblasts. Consistently, Irf7(-/-) mice are more vulnerable than Myd88(-/-) mice to viral infection, and this correlates with a marked decrease in serum IFN levels, indicating the importance of the IRF-7-dependent induction of systemic IFN responses for innate antiviral immunity. Furthermore, robust induction of IFN production by activation of the TLR9 subfamily in plasmacytoid dendritic cells is entirely dependent on IRF-7, and this MyD88-IRF-7 pathway governs the induction of CD8(+) T-cell responses. Thus, all elements of IFN responses, whether the systemic production of IFN in innate immunity or the local action of IFN from plasmacytoid dendritic cells in adaptive immunity, are under the control of IRF-7.
• Spatiotemporal regulation of MyD88-IRF-7 signalling for robust type-I interferon induction

  K Honda, Y Ohba, H Yanai, H Negishi, T Mizutani, A Takaoka, C Taya, T Taniguchi

  NATURE 434 (7036) 1035 - 1040 0028-0836 2005/04 [Refereed][Not invited]
   

  Robust type-I interferon (IFN-alpha/beta) induction in plasmacytoid dendritic cells, through the activation of Toll-like receptor 9 (TLR9), constitutes a critical aspect of immunity(1-6). It is absolutely dependent on the transcription factor IRF-7, which interacts with and is activated by the adaptor MyD88 (ref. 7). How plasmacytoid dendritic cells, but not other cell types (such as conventional dendritic cells), are able to activate the MyD88 IRF-7-dependent IFN induction pathway remains unknown. Here we show that the spatiotemporal regulation of MyD88 IRF-7 signalling is critical for a high-level IFN induction in response to TLR9 activation. The IFN-inducing TLR9 ligand, A/D-type CpG oligodeoxynucleotide (CpG-A)(3,4,8-11), is retained for long periods in the endosomal vesicles of plasmacytoid dendritic cells, together with the MyD88-IRF-7 complex. However, in conventional dendritic cells, CpG-A is rapidly transferred to lysosomal vesicles. We further show that conventional dendritic cells can also mount a robust IFN induction if CpG-A is manipulated for endosomal retention using a cationic lipid. This strategy also allows us to demonstrate endosomal activation of the IFN pathway by the otherwise inactive TLR9 ligand B/K-type oligodeoxynucleotide (CpG-B)(3,4,8-12). Thus, our study offers insights into the regulation of TLR9 signalling in space, potentially suggesting a new avenue for therapeutic intervention.
• Integral role of IRF-5 in the gene induction programme activated by Toll-like receptors

  A Takaoka, H Yanai, S Kondo, G Duncan, H Negishi, T Mizutani, S Kano, K Honda, Y Ohba, TW Mak, T Taniguchi

  NATURE 434 (7030) 243 - 249 0028-0836 2005/03 [Not refereed][Not invited]
   

  The activation of Toll-like receptors (TLRs) is central to innate and adaptive immunity(1-3). All TLRs use the adaptor MyD88 for signalling(4), but the mechanisms underlying the MyD88-mediated gene induction programme are as yet not fully understood. Here, we demonstrate that the transcription factor IRF-5 is generally involved downstream of the TLR-MyD88 signalling pathway for gene induction of proinflammatory cytokines, such as interleukin-6 (IL-6), IL-12 and tumour-necrosis factor-alpha. In haematopoietic cells from mice deficient in the Irf5 gene (Irf5(-/-) mice), the induction of these cytokines by various TLR ligands is severely impaired, whereas interferon-alpha induction is normal. We also provide evidence that IRF-5 interacts with and is activated by MyD88 and TRAF6, and that TLR activation results in the nuclear translocation of IRF-5 to activate cytokine gene transcription. Consistently, Irf5(-/-) mice show resistance to lethal shock induced by either unmethylated DNA or lipopolysaccharide, which correlates with a marked decrease in the serum levels of proinflammatory cytokines. Thus, our study identifies IRF-5 as a new, principal downstream regulator of the TLR-MyD88 signalling pathway and a potential target of therapeutic intervention to control harmful immune responses.
• Integral role of IRF-5 in the gene induction programme activated by Toll-like receptors

  A Takaoka, H Yanai, S Kondo, G Duncan, H Negishi, T Mizutani, S Kano, K Honda, Y Ohba, TW Mak, T Taniguchi

  NATURE 434 (7030) 243 - 249 0028-0836 2005/03 [Refereed][Not invited]
   

  The activation of Toll-like receptors (TLRs) is central to innate and adaptive immunity(1-3). All TLRs use the adaptor MyD88 for signalling(4), but the mechanisms underlying the MyD88-mediated gene induction programme are as yet not fully understood. Here, we demonstrate that the transcription factor IRF-5 is generally involved downstream of the TLR-MyD88 signalling pathway for gene induction of proinflammatory cytokines, such as interleukin-6 (IL-6), IL-12 and tumour-necrosis factor-alpha. In haematopoietic cells from mice deficient in the Irf5 gene (Irf5(-/-) mice), the induction of these cytokines by various TLR ligands is severely impaired, whereas interferon-alpha induction is normal. We also provide evidence that IRF-5 interacts with and is activated by MyD88 and TRAF6, and that TLR activation results in the nuclear translocation of IRF-5 to activate cytokine gene transcription. Consistently, Irf5(-/-) mice show resistance to lethal shock induced by either unmethylated DNA or lipopolysaccharide, which correlates with a marked decrease in the serum levels of proinflammatory cytokines. Thus, our study identifies IRF-5 as a new, principal downstream regulator of the TLR-MyD88 signalling pathway and a potential target of therapeutic intervention to control harmful immune responses.
• Negative regulation of Toll-like receptor signaling by IRF-4.

  NEGISHI Hideo, OHBA Yusuke, YANAI Hideyuki, TAKAOKA Akinori, HONMA Kiri, YUI Katsuyuki, MATSUYAMA Toshifumi, TANIGUCHI Tadatsugu, HONDA Kenya

  Natl. Acad. Sci. U.S.A. 102 (44) 15989 - 15994 0027-8424 2005 [Not refereed][Not invited]
  
• Role of a transductional-transcriptional processor complex involving MyD88 and IRF-7 in Toll-like receptor signaling

  K Honda, H Yanai, T Mizutani, H Negishi, N Shimada, N Suzuki, Y Ohba, A Takaoka, WC Yeh, T Taniguchi

  PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 101 (43) 15416 - 15421 0027-8424 2004/10 [Not refereed][Not invited]
   

  Toll-like receptor (TLR) activation is central to immunity, wherein the activation of the TLR9 subfamily members TLR9 and TLR7 results in the robust induction of type I IFNs (IFN-alpha/beta) by means of the MyD88 adaptor protein. However, it remains unknown how the TLR signal "input" can be processed through MyD88 to "output" the induction of the IFN genes. Here, we demonstrate that the transcription factor IRF-7 interacts with MyD88 to form a complex in the cytoplasm. We provide evidence that this complex also involves IRAK4 and TRAF6 and provides the foundation for the TLR9-dependent activation of the IFN genes. The complex defined in this study represents an example of how the coupling of the signaling adaptor and effector kinase molecules together with the transcription factor regulate the processing of an extracellular signal to evoke its versatile downstream transcriptional events in a cell. Thus, we propose that this molecular complex may function as a cytoplasmic transductional-transcriptional processor.
• Cell type-specific regulation of RhoA activity during cytokinesis

  H Yoshizaki, Y Ohba, MC Parrini, NG Dulyaninova, AR Bresnick, N Mochizuki, M Matsuda

  JOURNAL OF BIOLOGICAL CHEMISTRY 279 (43) 44756 - 44762 0021-9258 2004/10 [Not refereed][Not invited]
   

  Rho family GTPases play pivotal roles in cytokinesis. By using probes based on the principle of fluorescence resonance energy transfer (FRET), we have shown that in HeLa cells RhoA activity increases with the progression of cytokinesis. Here we show that in Rat1A cells RhoA activity remained suppressed during most of the cytokinesis. Consistent with this observation, the expression of C3 toxin inhibited cytokinesis in HeLa cells but not in Rat1A cells. Furthermore, the expression of a dominant negative mutant of Ect2, a Rho GEF, or Y-27632, an inhibitor of the Rho-dependent kinase ROCK, inhibited cytokinesis in HeLa cells but not in Rat1A cells. In contrast to the activity of RhoA, the activity of Rac1 was suppressed during cytokinesis and started increasing at the plasma membrane of polar sides before the abscission of the daughter cells in both HeLa and Rat1A cells. This type of Rac1 suppression was shown to be essential for cytokinesis because a constitutively active mutant of Rac1 induced a multinucleated phenotype in both HeLa and Rat1A cells. Moreover, the involvement of MgcRacGAP/CYK-4 in this suppression of Rac1 during cytokinesis was shown by the use of a dominant negative mutant. Because ML-7, an inhibitor of myosin light chain kinase, delayed the cytokinesis of Rat1A cells and because Pak, a Rac1 effector, is known to suppress myosin light chain kinase, the suppression of the Rac1-Pak pathway by MgcRacGAP may play a pivotal role in the cytokinesis of Rat1A cells.
• Cell type-specific regulation of RhoA activity during cytokinesis

  H Yoshizaki, Y Ohba, MC Parrini, NG Dulyaninova, AR Bresnick, N Mochizuki, M Matsuda

  JOURNAL OF BIOLOGICAL CHEMISTRY 279 (43) 44756 - 44762 0021-9258 2004/10 [Refereed][Not invited]
   

  Rho family GTPases play pivotal roles in cytokinesis. By using probes based on the principle of fluorescence resonance energy transfer (FRET), we have shown that in HeLa cells RhoA activity increases with the progression of cytokinesis. Here we show that in Rat1A cells RhoA activity remained suppressed during most of the cytokinesis. Consistent with this observation, the expression of C3 toxin inhibited cytokinesis in HeLa cells but not in Rat1A cells. Furthermore, the expression of a dominant negative mutant of Ect2, a Rho GEF, or Y-27632, an inhibitor of the Rho-dependent kinase ROCK, inhibited cytokinesis in HeLa cells but not in Rat1A cells. In contrast to the activity of RhoA, the activity of Rac1 was suppressed during cytokinesis and started increasing at the plasma membrane of polar sides before the abscission of the daughter cells in both HeLa and Rat1A cells. This type of Rac1 suppression was shown to be essential for cytokinesis because a constitutively active mutant of Rac1 induced a multinucleated phenotype in both HeLa and Rat1A cells. Moreover, the involvement of MgcRacGAP/CYK-4 in this suppression of Rac1 during cytokinesis was shown by the use of a dominant negative mutant. Because ML-7, an inhibitor of myosin light chain kinase, delayed the cytokinesis of Rat1A cells and because Pak, a Rac1 effector, is known to suppress myosin light chain kinase, the suppression of the Rac1-Pak pathway by MgcRacGAP may play a pivotal role in the cytokinesis of Rat1A cells.
• Role of a transductional-transcriptional processor complex involving MyD88 and IRF-7 in Toll-like receptor signaling

  K Honda, H Yanai, T Mizutani, H Negishi, N Shimada, N Suzuki, Y Ohba, A Takaoka, WC Yeh, T Taniguchi

  PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 101 (43) 15416 - 15421 0027-8424 2004/10 [Refereed][Not invited]
   

  Toll-like receptor (TLR) activation is central to immunity, wherein the activation of the TLR9 subfamily members TLR9 and TLR7 results in the robust induction of type I IFNs (IFN-alpha/beta) by means of the MyD88 adaptor protein. However, it remains unknown how the TLR signal "input" can be processed through MyD88 to "output" the induction of the IFN genes. Here, we demonstrate that the transcription factor IRF-7 interacts with MyD88 to form a complex in the cytoplasm. We provide evidence that this complex also involves IRAK4 and TRAF6 and provides the foundation for the TLR9-dependent activation of the IFN genes. The complex defined in this study represents an example of how the coupling of the signaling adaptor and effector kinase molecules together with the transcription factor regulate the processing of an extracellular signal to evoke its versatile downstream transcriptional events in a cell. Thus, we propose that this molecular complex may function as a cytoplasmic transductional-transcriptional processor.
• STAT3 noncell-autonomously controls planar cell polarity during zebrafish convergence and extension

  C Miyagi, S Yamashita, Y Ohba, H Yoshizaki, M Matsuda, T Hirano

  JOURNAL OF CELL BIOLOGY 166 (7) 975 - 981 0021-9525 2004/09 [Not refereed][Not invited]
   

  Zebrafish signal transducer and activator of transcription 3 (STAT3) controls the cell movements during gastrulation. Here, we show that noncell-autonomous activity of STAT3 signaling in gastrula organizer cells controls the polarity of neighboring cells through Dishevelled-RhoA signaling in the Wnt-planar cell polarity (WntPCP) pathway. In STAT3-depleted embryos, although all the known molecules in the Wnt-PCP pathway were expressed normally, the RhoA activity in lateral mesendodermal cells was down-regulated, resulting in severe cell polarization defects in convergence and extension movements identical to Strabismus-depleted embryos. Cell-autonomous activation of Wnt-PCP signaling by DeltaN-dishevelled rescued the defect in cell elongation, but not the orientation of lateral mesendodermal cells in STAT3-depleted embryos. The defect in the orientation could be rescued by transplantation of shield cells having noncellautonomous activity of STAT3 signaling. These results suggest that the cells undergoing convergence and extension movement may sense the gradient of signaling molecules, which are expressed in gastrula organizer by STAT3 and noncell-autonomously activate PCP signaling in neighboring cells during zebrafish gastrulation.
• STAT3 noncell-autonomously controls planar cell polarity during zebrafish convergence and extension

  C Miyagi, S Yamashita, Y Ohba, H Yoshizaki, M Matsuda, T Hirano

  JOURNAL OF CELL BIOLOGY 166 (7) 975 - 981 0021-9525 2004/09 [Refereed][Not invited]
   

  Zebrafish signal transducer and activator of transcription 3 (STAT3) controls the cell movements during gastrulation. Here, we show that noncell-autonomous activity of STAT3 signaling in gastrula organizer cells controls the polarity of neighboring cells through Dishevelled-RhoA signaling in the Wnt-planar cell polarity (WntPCP) pathway. In STAT3-depleted embryos, although all the known molecules in the Wnt-PCP pathway were expressed normally, the RhoA activity in lateral mesendodermal cells was down-regulated, resulting in severe cell polarization defects in convergence and extension movements identical to Strabismus-depleted embryos. Cell-autonomous activation of Wnt-PCP signaling by DeltaN-dishevelled rescued the defect in cell elongation, but not the orientation of lateral mesendodermal cells in STAT3-depleted embryos. The defect in the orientation could be rescued by transplantation of shield cells having noncellautonomous activity of STAT3 signaling. These results suggest that the cells undergoing convergence and extension movement may sense the gradient of signaling molecules, which are expressed in gastrula organizer by STAT3 and noncell-autonomously activate PCP signaling in neighboring cells during zebrafish gastrulation.
• RalA activation at nascent lamellipodia of epidermal growth factor-stimulated Cos7 cells and migrating Madin-Darby canine kidney Cells

  A Takaya, Y Ohba, K Kurokawa, M Matsuda

  MOLECULAR BIOLOGY OF THE CELL 15 (6) 2549 - 2557 1059-1524 2004/06 [Not refereed][Not invited]
   

  Ra1A, a member of the Ras-family GTPases, regulates various cellular functions such as filopodia formation, endocytosis, and exocytosis. On epidermal growth factor (EGF) stimulation, activated Ras recruits guanine nucleotide exchange factors (GEFs) for RalA, followed by RalA activation. By using fluorescence resonance energy transfer-based probes for RalA activity, we found that the EGF-induced RalA activation in Cos7 cells was restricted at the EGF-induced nascent lamellipodia, whereas under a similar condition both Ras activation and Ras-dependent translocation of Ral GEFs occurred more diffusely at the plasma membrane. This EGF-induced RalA activation was not observed when lamellipodial protrusion was suppressed by a dominant negative mutant of Rac1, a GTPase-activating protein for Cdc42, inhibitors of phosphatidylinositol 3-kinase, or inhibitors of actin polymerization. On the other hand, EGF-induced lamellipodial protrusion was inhibited by microinjection of the RalA-binding domains of Ra1BP1 and Sec5. Furthermore, we found that RalA activity was high at the lamellipodia of migrating Madin-Darby canine kidney cells and that the migration of Madin-Darby canine kidney cells was perturbed by the microinjection of RalBP1-RalA-binding domain. Thus, RalA activation is required for the induction of lamellipodia, and conversely, lamellipodial protrusion seems to be required for the RalA activation, suggesting the presence of a positive feedback loop between RalA activation and lamellipodial protrusion. Our observation also demonstrates that the spatial regulation of RalA is conducted by a mechanism distinct from the temporal regulation conducted by Ras-dependent plasma membrane recruitment of Ral guanine nucleotide exchange factors.
• RalA activation at nascent lamellipodia of epidermal growth factor-stimulated Cos7 cells and migrating Madin-Darby canine kidney Cells

  A Takaya, Y Ohba, K Kurokawa, M Matsuda

  MOLECULAR BIOLOGY OF THE CELL 15 (6) 2549 - 2557 1059-1524 2004/06 [Refereed][Not invited]
   

  Ra1A, a member of the Ras-family GTPases, regulates various cellular functions such as filopodia formation, endocytosis, and exocytosis. On epidermal growth factor (EGF) stimulation, activated Ras recruits guanine nucleotide exchange factors (GEFs) for RalA, followed by RalA activation. By using fluorescence resonance energy transfer-based probes for RalA activity, we found that the EGF-induced RalA activation in Cos7 cells was restricted at the EGF-induced nascent lamellipodia, whereas under a similar condition both Ras activation and Ras-dependent translocation of Ral GEFs occurred more diffusely at the plasma membrane. This EGF-induced RalA activation was not observed when lamellipodial protrusion was suppressed by a dominant negative mutant of Rac1, a GTPase-activating protein for Cdc42, inhibitors of phosphatidylinositol 3-kinase, or inhibitors of actin polymerization. On the other hand, EGF-induced lamellipodial protrusion was inhibited by microinjection of the RalA-binding domains of Ra1BP1 and Sec5. Furthermore, we found that RalA activity was high at the lamellipodia of migrating Madin-Darby canine kidney cells and that the migration of Madin-Darby canine kidney cells was perturbed by the microinjection of RalBP1-RalA-binding domain. Thus, RalA activation is required for the induction of lamellipodia, and conversely, lamellipodial protrusion seems to be required for the RalA activation, suggesting the presence of a positive feedback loop between RalA activation and lamellipodial protrusion. Our observation also demonstrates that the spatial regulation of RalA is conducted by a mechanism distinct from the temporal regulation conducted by Ras-dependent plasma membrane recruitment of Ral guanine nucleotide exchange factors.
• Coactivation of Rad1 and Cdc42 at lamellipodia and membrane ruffles induced by epidermal growth factor

  K Kurokawa, RE Itoh, H Yoshizaki, Y Ohba, T Nakamura, M Matsuda

  MOLECULAR BIOLOGY OF THE CELL 15 (3) 1003 - 1010 1059-1524 2004/03 [Not refereed][Not invited]
   

  A major function of Rho-family GTPases is to regulate the organization of the actin cytoskeleton; filopodia, lamellipodia, and stress fiber are regarded as typical phenotypes of the activated Cdc42, Rac, and Rho, respectively. Using probes based on fluorescent resonance energy transfer, we report on the spatiotemporal regulation of Rac1 and Cdc42 at lamellipodia and membrane ruffles. In epidermal growth factor (EGF)-stimulated Cos1 and A431 cells, both Rac1 and Cdc42 were activated diffusely at the plasma membrane, followed by lamellipodial protrusion and membrane ruffling. Although Rac1 activity subsided rapidly, Cdc42 activity was sustained at lamellipodia. A critical role of Cdc42 in these EGF-induced morphological changes was demonstrated as follows. First, phorbol 12-myristate 13-acetate, which activated Rac1 but not Cdc42, could not induce full-grown lamellipodia in Cos1 cells. Second, a GTPase-activating protein for Cdc42, KIAA1204/ CdGAP, inhibited lamellipodial protrusion and membrane ruffling without interfering with Rac1 activation. Third, expression of the Cdc42-binding domain of N-WASP inhibited the EGF-induced morphological changes. Therefore, Rac1 and Cdc42 seem to synergistically induce lamellipodia and membrane ruffles in EGF-stimulated Cos1 cells and A431 cells.
• Visualization of signal transduction of Ras

  Y Ohba, M Matsuda

  SEIKAGAKU 76 (1) 16 - 28 0037-1017 2004/01 [Refereed][Not invited]
  
• Activity of Rho-family GTPases during cell division as visualized with FRET-based probes

  H Yoshizaki, Y Ohba, K Kurokawa, RE Itoh, T Nakamura, N Mochizuki, K Nagashima, M Matsuda

  JOURNAL OF CELL BIOLOGY 162 (2) 223 - 232 0021-9525 2003/07 [Not refereed][Not invited]
   

  Rho-family GTPases regulate many cellular functions. To visualize the activity of Rho-family GTPases in living cells, we developed fluorescence resonance energy transfer (FRET)-based probes for Rac1 and Cdc42 previously (Itoh, R.E., K. Kurokawa, Y. Ohba, H. Yoshizaki, N. Mochizuki, and M. Matsuda. 2002. Mol. Cell. Biol. 22:6582-6591). Here, we added two types of probes for RhoA. One is to monitor the activity balance between guanine nucleotide exchange factors and GTPase-activating proteins, and another is to monitor the level of GTP-RhoA. Using these FRET probes, we imaged the activities of Rho-family GTPases during the cell division of HeLa cells. The activities of RhoA, Rac1, and Cdc42 were high at the plasma membrane in interphase, and decreased rapidly on entry into M phase. From after anaphase, the RhoA activity increased at the plasma membrane including cleavage furrow. Rac1 activity was suppressed at the spindle midzone and increased at the plasma membrane of polar sides after telophase. Cdc42 activity was suppressed at the plasma membrane and was high at the intracellular membrane compartments during cytokinesis. In conclusion, we could use the FRET-based probes to visualize the complex spatio-temporal regulation of Rho-family GTPases during cell division.
• Activity of Rho-family GTPases during cell division as visualized with FRET-based probes

  H Yoshizaki, Y Ohba, K Kurokawa, RE Itoh, T Nakamura, N Mochizuki, K Nagashima, M Matsuda

  JOURNAL OF CELL BIOLOGY 162 (2) 223 - 232 0021-9525 2003/07 [Refereed][Not invited]
   

  Rho-family GTPases regulate many cellular functions. To visualize the activity of Rho-family GTPases in living cells, we developed fluorescence resonance energy transfer (FRET)-based probes for Rac1 and Cdc42 previously (Itoh, R.E., K. Kurokawa, Y. Ohba, H. Yoshizaki, N. Mochizuki, and M. Matsuda. 2002. Mol. Cell. Biol. 22:6582-6591). Here, we added two types of probes for RhoA. One is to monitor the activity balance between guanine nucleotide exchange factors and GTPase-activating proteins, and another is to monitor the level of GTP-RhoA. Using these FRET probes, we imaged the activities of Rho-family GTPases during the cell division of HeLa cells. The activities of RhoA, Rac1, and Cdc42 were high at the plasma membrane in interphase, and decreased rapidly on entry into M phase. From after anaphase, the RhoA activity increased at the plasma membrane including cleavage furrow. Rac1 activity was suppressed at the spindle midzone and increased at the plasma membrane of polar sides after telophase. Cdc42 activity was suppressed at the plasma membrane and was high at the intracellular membrane compartments during cytokinesis. In conclusion, we could use the FRET-based probes to visualize the complex spatio-temporal regulation of Rho-family GTPases during cell division.
• Mechanism of the spatio-temporal regulation of Ras and Rap1

  Y Ohba, K Kurokawa, M Matsuda

  EMBO JOURNAL 22 (4) 859 - 869 0261-4189 2003/02 [Not refereed][Not invited]
   

  Epidermal growth factor (EGF) activates Ras and Rap1 at distinct intracellular regions. Here, we explored the mechanism underlying this phenomenon. We originally noticed that in cells expressing Epac, a cAMP-dependent Rap1 GEF (guanine nucleotide exchange factor), cAMP activated Rap1 at the perinuclear region, as did EGF. However, in cells expressing e-GRF, a recombinant cAMP-responsive Ras GEF, cAMP activated Ras at the peripheral plasma membrane. Based on the uniform cytoplasmic expression of Epac and e-GRF, GEF did not appear to account for the non-uniform increase in the activities of Ras and Rap1. In contrast, when we used probes with reduced sensitivity to GTPase-activating proteins (GAPs), both Ras and Rap1 appeared to be activated uniformly in the EGF-stimulated cells. Furthermore, we calculated the local rate constants of GEFs and GAPs from the video images of Ras activation and found that GAP activity was higher at the central plasma membrane than the periphery. Thus we propose that GAP primarily dictates the spatial regulation of Ras family G proteins, whereas GEF primarily determines the timing of Ras activation.
• Mechanism of the spatio-temporal regulation of Ras and Rap1

  Y Ohba, K Kurokawa, M Matsuda

  EMBO JOURNAL 22 (4) 859 - 869 0261-4189 2003/02 [Refereed][Not invited]
   

  Epidermal growth factor (EGF) activates Ras and Rap1 at distinct intracellular regions. Here, we explored the mechanism underlying this phenomenon. We originally noticed that in cells expressing Epac, a cAMP-dependent Rap1 GEF (guanine nucleotide exchange factor), cAMP activated Rap1 at the perinuclear region, as did EGF. However, in cells expressing e-GRF, a recombinant cAMP-responsive Ras GEF, cAMP activated Ras at the peripheral plasma membrane. Based on the uniform cytoplasmic expression of Epac and e-GRF, GEF did not appear to account for the non-uniform increase in the activities of Ras and Rap1. In contrast, when we used probes with reduced sensitivity to GTPase-activating proteins (GAPs), both Ras and Rap1 appeared to be activated uniformly in the EGF-stimulated cells. Furthermore, we calculated the local rate constants of GEFs and GAPs from the video images of Ras activation and found that GAP activity was higher at the central plasma membrane than the periphery. Thus we propose that GAP primarily dictates the spatial regulation of Ras family G proteins, whereas GEF primarily determines the timing of Ras activation.
• Novel function of Chat in controlling cell adhesion via Cas-Crk-C3G-pathway-mediated Rap1 activation

  A Sakakibara, Y Ohba, K Kurokawa, M Matsuda, S Hattori

  JOURNAL OF CELL SCIENCE 115 (24) 4915 - 4924 0021-9533 2002/12 [Not refereed][Not invited]
   

  Chat (Cas/HEF1-associated signal transducer) is a novel signaling molecule with an N-terminal SH2 domain and C-terminal Cas/HEF1 association domain that is implicated in the regulation of cell adhesion. The Cas/HEF1 association domain also shows sequence similarity with guanine nucleotide exchange factors for Ras family small GTPases. In this study, we found significant activation of Rap1 in Chat-overexpressing cells. Myr-Chat, a membrane-targeted form of Chat, activated Rap1 more efficiently. Interestingly, Chat and Cas synergistically activated Rap1. Certain Cas, Crk or C3G mutants suppressed Rap I activation by Chat. We also confirmed the ternary complex formation consisting of Chat, Cas and Crk. Thus, it is likely that Chat-induced Rap1 activation was mediated by upregulation of the Cas-Crk-C3G signaling pathway rather than direct guanine nucleotide exchange factor activity of Chat. We further demonstrated that Myr-Chat expression induced cell periphery spreading and cell shape branching and that this activity also depended on the Cas-Crk-C3G pathway and Rap1 activity. Moreover, expression of Myr-Chat enhanced integrin-mediated cell adhesion. Taken together we propose a novel role for the Chat-Cas complex in controlling cell adhesion via the activation of Rap1.
• Novel function of Chat in controlling cell adhesion via Cas-Crk-C3G-pathway-mediated Rap1 activation

  A Sakakibara, Y Ohba, K Kurokawa, M Matsuda, S Hattori

  JOURNAL OF CELL SCIENCE 115 (24) 4915 - 4924 0021-9533 2002/12 [Refereed][Not invited]
   

  Chat (Cas/HEF1-associated signal transducer) is a novel signaling molecule with an N-terminal SH2 domain and C-terminal Cas/HEF1 association domain that is implicated in the regulation of cell adhesion. The Cas/HEF1 association domain also shows sequence similarity with guanine nucleotide exchange factors for Ras family small GTPases. In this study, we found significant activation of Rap1 in Chat-overexpressing cells. Myr-Chat, a membrane-targeted form of Chat, activated Rap1 more efficiently. Interestingly, Chat and Cas synergistically activated Rap1. Certain Cas, Crk or C3G mutants suppressed Rap I activation by Chat. We also confirmed the ternary complex formation consisting of Chat, Cas and Crk. Thus, it is likely that Chat-induced Rap1 activation was mediated by upregulation of the Cas-Crk-C3G signaling pathway rather than direct guanine nucleotide exchange factor activity of Chat. We further demonstrated that Myr-Chat expression induced cell periphery spreading and cell shape branching and that this activity also depended on the Cas-Crk-C3G pathway and Rap1 activity. Moreover, expression of Myr-Chat enhanced integrin-mediated cell adhesion. Taken together we propose a novel role for the Chat-Cas complex in controlling cell adhesion via the activation of Rap1.
• Activation of Rac and Cdc42 video imaged by fluorescent resonance energy transfer-based single-molecule probes in the membrane of living cells

  RE Itoh, K Kurokawa, Y Ohba, H Yoshizaki, N Mochizuki, M Matsuda

  MOLECULAR AND CELLULAR BIOLOGY 22 (18) 6582 - 6591 0270-7306 2002/09 [Not refereed][Not invited]
   

  Rho family G proteins, including Rac and Cdc42, regulate a variety of cellular functions such as morphology, motility, and gene expression. We developed fluorescent resonance energy transfer-based probes which monitored the local balance between the activities of guanine nucleotide exchange factors and GTPase-activating proteins for Rac1 and Cdc42 at the membrane. These probes, named Raichu-Rac and Raichu-Cdc42, consisted of a Cdc42- and Rac-binding domain of Pak, Rac1 or Cdc42, a pair of green fluorescent protein mutants, and a CAAX box of Ki-Ras. With these probes, we video imaged the Rac and Cdc42 activities. In motile HT1080 cells, activities of both Rac and Cdc42 gradually increased toward the leading edge and decreased rapidly when cells changed direction. Under a higher magnification, we observed that Rac activity was highest immediately behind the leading edge, whereas Cdc42 activity was most prominent at the tip of the leading edge. Raichu-Rac and Raichu-Cdc42 were also applied to a rapid and simple assay for the analysis of putative guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs) in living cells. Among six putative GEFs and GAPS, we identified KIAA0362/DBS as a GEF for Rac and Cdc42, KIAA1256 as a GEF for Cdc42, KIAA0053 as a GAP for Rac and Cdc42, and KIAA1204 as a GAP for Cdc42. In conclusion, use of these single-molecule probes to determine Rac and Cdc42 activity will accelerate the analysis of the spatiotemporal regulation of Rac and Cdc42 in a living cell.
• Activation of Rac and Cdc42 video imaged by fluorescent resonance energy transfer-based single-molecule probes in the membrane of living cells

  RE Itoh, K Kurokawa, Y Ohba, H Yoshizaki, N Mochizuki, M Matsuda

  MOLECULAR AND CELLULAR BIOLOGY 22 (18) 6582 - 6591 0270-7306 2002/09 [Refereed][Not invited]
   

  Rho family G proteins, including Rac and Cdc42, regulate a variety of cellular functions such as morphology, motility, and gene expression. We developed fluorescent resonance energy transfer-based probes which monitored the local balance between the activities of guanine nucleotide exchange factors and GTPase-activating proteins for Rac1 and Cdc42 at the membrane. These probes, named Raichu-Rac and Raichu-Cdc42, consisted of a Cdc42- and Rac-binding domain of Pak, Rac1 or Cdc42, a pair of green fluorescent protein mutants, and a CAAX box of Ki-Ras. With these probes, we video imaged the Rac and Cdc42 activities. In motile HT1080 cells, activities of both Rac and Cdc42 gradually increased toward the leading edge and decreased rapidly when cells changed direction. Under a higher magnification, we observed that Rac activity was highest immediately behind the leading edge, whereas Cdc42 activity was most prominent at the tip of the leading edge. Raichu-Rac and Raichu-Cdc42 were also applied to a rapid and simple assay for the analysis of putative guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs) in living cells. Among six putative GEFs and GAPS, we identified KIAA0362/DBS as a GEF for Rac and Cdc42, KIAA1256 as a GEF for Cdc42, KIAA0053 as a GAP for Rac and Cdc42, and KIAA1204 as a GAP for Cdc42. In conclusion, use of these single-molecule probes to determine Rac and Cdc42 activity will accelerate the analysis of the spatiotemporal regulation of Rac and Cdc42 in a living cell.
• A pair of fluorescent resonance energy transfer-based probes for tyrosine phosphorylation of the CrkII adaptor protein in vivo

  K Kurokawa, N Mochizuki, Y Ohba, H Mizuno, A Miyawaki, M Matsuda

  JOURNAL OF BIOLOGICAL CHEMISTRY 276 (33) 31305 - 31310 0021-9258 2001/08 [Refereed][Not invited]
   

  An adaptor protein, CrkII, which is involved in a variety of signaling cascades such as cell growth, migration, and apoptosis, becomes phosphorylated on Tyr.. upon stimulation. Here, we report on a fluorescent resonance energy transfer-based sensor, which consists of CrkII sandwiched with cyan- and yellow-emitting variants of green fluorescent protein. This protein enabled us to monitor rapid and transient phosphorylation of CrkII upon epidermal growth factor stimulation in a living cell. However, rapid diffusion of the probes prevented us from specifying where the phosphorylation started within the cell. To overcome this problem, we fused the CAAX box of Ki-Ras to the carboxyl terminus of this probe and restricted its localization mostly to the plasma membrane. With this modified probe, we found that epidermal growth factor-induced phosphorylation of CrkII was initiated at the peripheral plasma membrane, moving toward the center of the cell. Moreover, this CAAX box-fused probe showed improvement in sensitivity and time resolution of the monitoring of CrkII phosphorylation. Thus, this pair of CrkII probes visualizes dynamic changes in the total and local levels of the tyrosine phosphorylation of CrkII in a living cell.
• Requirement for C3G-dependent Rap1 activation for cell adhesion and embryogenesis

  Y Ohba, K Ikuta, A Ogura, J Matsuda, N Mochizuki, K Nagashima, K Kurokawa, BJ Mayer, K Maki, J Miyazaki, M Matsuda

  EMBO JOURNAL 20 (13) 3333 - 3341 0261-4189 2001/07 [Not refereed][Not invited]
   

  C3G is a guanine nucleotide exchange factor (GEF) for Rap1, and is activated via Crk adaptor protein. To understand the physiological role of C3G, we generated C3G knockout mice. C3G(-/-) homozygous mice died before embryonic day 7.5. The lethality was rescued by the expression of the human C3G transgene, which could be excised upon the expression of Cre recombinase, From the embryo of this mouse, we prepared fibroblast cell lines, MEF-hC3G. Expression of Cre abolished the expression of C3G in MEF-hC3G and inhibited cell adhesion-induced activation of Rap1. The Cre-expressing MEF-hC3G showed impaired cell adhesion, delayed cell spreading and accelerated cell migration. The accelerated cell migration was suppressed by the expression of active Rap1, Rap2 and R-Ras, Expression of Epac and CalDAG-GEFI, GEFs for Rap1, also suppressed the accelerated migration of the CSG-deficient cells. This observation indicated that Rap1 activation was sufficient to complement the C3G deficiency. In conclusion, C3G-dependent activation of Rap1 is required for adhesion and spreading of embryonic fibroblasts and for the early embryogenesis of the mouse.
• Spatio-temporal images of growth-factor-induced activation of Ras and Rap1

  N Mochizuki, S Yamashita, K Kurokawa, Y Ohba, T Nagai, A Miyawaki, M Matsuda

  NATURE 411 (6841) 1065 - 1068 0028-0836 2001/06 [Not refereed][Not invited]
   

  G proteins of the Ras family function as molecular switches in many signalling cascades(1-3); however, little is known about where they become activated in living cells. Here we use FRET (fluorescent resonance energy transfer)-based sensors to report on the spatio-temporal images of growth-factor-induced activation of Ras and Rap1. Epidermal growth factor activated Ras at the peripheral plasma membrane and Rap1 at the intracellular perinuclear region of COS-1 cells. In PC12 cells, nerve growth factor-induced activation of Ras was initiated at the plasma membrane and transmitted to the whole cell body. After three hours, high Ras activity was observed at the extending neurites. By using the FRAP (fluorescence recovery after photobleaching) technique, we found that Ras at the neurites turned over rapidly; therefore, the sustained Ras activity at neurites was due to high GTP/GDP exchange rate and/or low GTPase activity, but not to the retention of the active Ras. These observations may resolve long-standing questions as to how Ras and Rap1 induce different cellular responses(4) and how the signals for differentiation and survival are distinguished by neuronal cells(5).
• CalDAG-GEFIII activation of Ras, R-Ras, and Rap1

  S Yamashita, N Mochizuki, Y Ohba, M Tobiume, Y Okada, H Sawa, K Nagashima, M Matsuda

  JOURNAL OF BIOLOGICAL CHEMISTRY 275 (33) 25488 - 25493 0021-9258 2000/08 [Not refereed][Not invited]
   

  We characterized a novel guanine nucleotide exchange factor (GEF) for Ras family G proteins that is highly homologous to CalDAG-GEFI, a GEF for Rap1 and R-Ras, and to RasGRP/CalDAG-GEFII, a GEF for Res and R-Ras. This novel GEF, referred to as CalDAG-GEFIII, increased the GTP/GDP ratio of Ha-Ras, R-Ras, and Rap1 in 293T cells. CalDAG-GEFIII promoted the guanine nucleotide exchange of Ha-Ras, R-Ras, and Rap1 in vitro also, indicating that CalDAG-GEFIII exhibited the widest substrate specificity among the known GEFs for Res family G proteins. Expression of CalDAG-GEFIII was detected in the glial cells of the brain and the glomerular mesangial cells of the kidney by in. situ hybridization. CalDAG-GEFIII activated ERW MAPK most efficiently, followed by CalDAG-GEFII and CalDAG-GEFI in 293T cells. JNK activation was most prominent in cells expressing CalDAG-GEFII, followed by CalDAG-GEFIII and CaLDAG-GEFI. Expression of CalDAG-GEFIII induced neuronal differentiation of PC12 cells and anchorage-independent growth of Rat1A cells less efficiently than did CalDAG-GEFII. Thus, coactivation of Rap1 by CalDAG-GEFIII apparently attenuated Ras-MAPK-dependent neuronal differentiation and cellular transformation. Altogether, CalDAG-GEFIII activated a broad range of Res family Gr proteins and exhibited a biological activity different from that of either CalDAG-GEFI or CalDAG-GEFII.
• Rap2 as a slowly responding molecular switch in the Rap1 signaling cascade

  Y Ohba, N Mochizuki, K Matsuo, S Yamashita, M Nakaya, Y Hashimoto, M Hamaguchi, T Kurata, K Nagashima, M Matsuda

  MOLECULAR AND CELLULAR BIOLOGY 20 (16) 6074 - 6083 0270-7306 2000/08 [Not refereed][Not invited]
   

  Rap2 is a member of the Ras family of GTPases and exhibits 60% identity to Rap1, but the function and regulation of Rap2 remain obscure. We found that, unlike the other Ras family proteins, the GTP-bound active form exceeded 50% of total Rap2 protein in adherent cells. Guanine nucleotide exchange factors (GEFs) for Rap1, C3G, Epac (or cyclic AMP [cAMP]-GEF), CalDAG-GEFI, PDZ-GEF1, and GFR efficiently increased the level of GTP-Rap2 both in 293T cells and in vitro. GTPase-activating proteins (GAPs) for Rap1, rap1GAPII and SPA-1, stimulated Rap2 GTPase, but with low efficiency. The half-life of GTP-Rap2 was significantly longer than that of GTP-Rap1 in 293T cells, indicating that low sensitivity to GAPs caused a high GTP/GDP ratio on Rap2. Rap2 bound to the Ras-binding domain of Raf and inhibited Ras dependent activation of Elk1 transcription factor, as did Rap1. The level of GTP-Rap2 in rat 3Y1 fibroblasts was decreased by the expression of v-Src, and expression of a GTPase-deficient Rap2 mutant inhibited v-Src-dependent transformation of 3Y1 cells. Altogether, Rap2 is regulated by a similar set of GEFs and GAPs as Rap1 and functions as a slowly responding molecular switch in the Rap1 signaling cascade.
• Regulatory proteins of R-Ras, TC21/R-Ras2, and M-Ras/R-Ras3

  Y Ohba, N Mochizuki, S Yamashita, AM Chan, JW Schrader, S Hattori, K Nagashima, M Matsuda

  JOURNAL OF BIOLOGICAL CHEMISTRY 275 (26) 20020 - 20026 0021-9258 2000/06 [Not refereed][Not invited]
   

  We studied the regulation of three closely related members of Pas family G proteins, R-Ras, TC21 (also known as R-Ras2), and M-Ras (R-Ras3). Guanine nucleotide exchange of R-Ras and TC21 was promoted by RasGRF, C3G, CalDAG-GEFI, CalDAG-GEFII (RasGRP), and CalDAG-GEFIII both in 293T cells and in vitro. By contrast, guanine nucleotide exchange of M-Ras was promoted by the guanine nucleotide exchange factors (GEFs) for the classical Pas (Ha-, K-, and N-), including mSos, RasGRF, CalDAG-GEFII, and CalDAG-GEFIII. GTPase-activating proteins (GAPs) for Pas, Gap1(m), p120 GAP, and NF-1 stimulated all of the R-Ras, TC21, and M-Ras proteins, whereas R-Ras GAP stimulated R-Ras and TC21 but not M-Ras. We did not find any remarkable difference in the subcellular localization of R-Ras, TC21, or M-Ras when these were expressed with a green fluorescent protein tag in 293T cells and MDCK cells. In conclusion, TC21 and R-Ras were regulated by the same GEFs and GAPs, whereas M-Ras was regulated as the classical Ras.
• Crk activation of JNK via C3G and R-Ras

  N Mochizuki, Y Ohba, S Kobayashi, N Otsuka, AM Graybiel, S Tanaka, M Matsuda

  JOURNAL OF BIOLOGICAL CHEMISTRY 275 (17) 12667 - 12671 0021-9258 2000/04 [Not refereed][Not invited]
   

  v-crk is an oncogene identified originally in CT10 chicken tumor virus. C3G, a guanine nucleotide exchange factor (GEF) for Rap1 and R-Ras, is postulated to transduce the oncogenic signal of v-Crk to c-Jun kinase (JNK). We have found that R-Ras, but not Rap1, mediates JNK activation by v-Crk in 293T and NIH 3T3 cells. Constitutively activated R-Ras, R-Ras(Val-38), but not Rap1(Val-12) activated JNK, as did the constitutively active H-Ras(Val-12) or Bac1(Val-12). v-Crk activation of JNK was inhibited by a dominant-negative mutant of R-Ras, R-Ras(Asn-43). JNK activation by R-Ras(Val-38) was inhibited by a dominant-negative mutant of mixed lineage kinase 3. Among six GEFs for Res-family G proteins, mSos1, Ras-GRF, C3G, CalDAG-GEFI, Ras-GRP/CalDAG-GEFII, and Epac/cAMP-GEFI, GEFs for either H-Ras or R-Ras activated JNK and c-Jun-dependent transcription. CalDAG-GEFI and Epac/cAMP-GEFI, both of which are GEFs specific for Rap1, did not activate JNK or c-Jun-dependent transcription. These results demonstrate that R-Ras, but not Rap1, is the downstream effector of C3G to stimulate JNK, Finally, we found that expression of the dominant-negative R-Ras mutant induced flat reversion of NIH 3T3 cells transformed by v-Crk, suggesting that R-Ras-dependent JNK activation is critical for the transformation by v-Crk.
• Eosinophilic inclusions in ependymoma represent microlumina: a light and electron microscopic study

  N Kawano, Y Ohba, K Nagashima

  ACTA NEUROPATHOLOGICA 99 (2) 214 - 218 0001-6322 2000/02 [Not refereed][Not invited]
   

  A study was undertaken to determine the pathological significance of previously unrecognized intracytoplasmic eosinophilic inclusions (IEIs) in ependymoma. The study group consisted of 58 ependymomas, all of which were pathologically characterized and graded according to the 1993 WHO classification. Electron microscopic studies were performed in 16 cases. The study showed that 33 (57%) ependymomas had IEIs and that in 8 cases these were abundant. Round and eosinophilic, their sizes varied from 10 mu m to a tiny dot. Similar eosinophilic bodies were also observed between tumor cells. The inclusions were weakly PAS positive. On immunostains, IEIs were frequently positive for glial fibrillary acidic protein, less often for S-100 protein, and for epithelial membrane protein and CAM 5.2. They were negative for AE1/AE3, carcinoembryonic antigen and Ber-EP4. Ultrastructurally, IEIs represented intracytoplasmic lumens containing microvilli and cilia. These microlumina also frequently contained granule-tubular materials. With reference to tumor subtypes, IEIs occurred most frequently in ordinary and clear cell ependymomas. IEIs were also present in 4 of 6 anaplastic ependymomas studied. In conclusion, IEIs represent microlumina and occur in more than a half of ependymomas including malignant examples. Their finding is a helpful diagnostic feature of ependymoma.
• A pair of fluorescent resonance energy transfer-based probes for tyrosine phosphorylation of the CrkII adaptor protein in vivo.

  J. Biol. Chem. 276 (33) 6074 - 6083 2000 [Not refereed][Not invited]
  
• Activation of the ERK/MAPK pathway by an isoform of rap1GAP associated with G alpha(i)

  N Mochizuki, Y Ohba, E Kiyokawa, T Kurata, T Murakami, T Ozaki, A Kitabatake, K Nagashima, M Matsuda

  NATURE 400 (6747) 891 - 894 0028-0836 1999/08 [Not refereed][Not invited]
   

  Many receptors for neuropeptides and hormones are coupled with the heterotrimeric G(i) protein, which activates the p42/44 mitogen-activated protein kinase (ERK/MAPE;) cascade through both the alpha- and beta gamma-subunits of G(i) (refs 1-3). The beta gamma-subunit activates the ERK/MAPK cascade through tyrosine kinase(4-6). Constitutively active G alpha(i2) (gip2) isolated from adrenal and ovarian tumours(7,8) transforms Rat-1 fibroblasts and also activates the ERK/MAPK cascade by an unknown mechanism(9,10). The ERK/MAPK pathway is activated by Ras, and is inhibited when the low-molecular-mass GTP-binding protein Rap1 antagonizes pas function(11). Here we show that a novel isoform of Rap1 GTPase-activating protein, called rap1GAPII, binds specifically to the alpha-subunits of the G(i) family of heterotrimeric G-proteins. Stimulation of the G(i)-coupled m2-muscarinic receptor translocates rap1GAPII from the cytosol to the membrane and decreases the amount of GTP-bound Rap1. This decrease in GTP-bound Rap1 activates ERK/MAPK. Thus, the alpha-subunit of G(i) activates the Ras-ERK/MAPK mitogenic pathway by membrane recruitment of rap1GAPII and reduction of GTP-bound Rap1.
• Melanotic peritoneal sarcomatosis originating from clear cell sarcoma

  Yusuke Ohba, Hiroaki Suzuki, Hiroaki Hiraga, Tomoo Ito, Hirofumi Sawa, Makoto Nagai, Shin-Ichi Satoh, Hiroyuki Iwaki, Kazuo Nagashima

  Pathology International 49 (7) 653 - 657 1320-5463 1999 [Not refereed][Not invited]
   

  Peritoneal sarcomatosis was found in a 53-year-old male who had a history of resection of clear cell sarcoma (CCS) of the right wrist 7 years previously. Both the previous wrist tumor and the peritoneal disseminants consisted of small, spindle-shaped cells occasionally containing melanophages. Histologic features, histochemical demonstration of argentaffin granules, immunohistochemical reaction with HMB 45, and the demonstration of a chimeric transcript of EWS-ATF-1 established the diagnosis of CCS in the peritoneal tumors. As far as we are aware, this is the first case of a peritoneal sarcomatosis associated with CCS.

MISC

• アレナウイルスZ蛋白の自己重合を検出するFRETバイオセンサーの開発と,それを利用した抗ウイルス薬スクリーニング法の確立

  水谷龍明, 大場雄介, 水田賢志, 浦田秀造  日本蛋白質科学会年会プログラム・要旨集  23rd (CD-ROM)-  2023
• フラビウイルスの粒子産生における分泌型NS1タンパク質の役割

  田村友和, 田村友和, 鳥居志保, 鳥居志保, 梶原健太郎, 安齋樹, 藤岡容一朗, 野田暉翔, 田鍬修平, 森岡佑平, 森岡佑平, 鈴木理滋, YUZY Fauzyah, 小野慎子, 小野慎子, 大場雄介, 岡田雅人, 福原崇介, 福原崇介, 松浦善治, 松浦善治  日本ウイルス学会学術集会プログラム・予稿集(Web)  69th-  2022
• Visualizing virus entry into host cells

  藤岡容一朗, 天野麻穂, 大場雄介  医学のあゆみ  280-  (9)  2022
• Pathogen dynamics in host cells

  藤岡容一朗, 天野麻穂, 大場雄介  実験医学  39-  (2)  2021
• エボラウイルスエンベロープへのホスファチジルセリン集積に関与する宿主スクランブラーゼの同定

  南保 明日香, 丸山 隼輝, 今井 正樹, 氏江 美智子, 高田 礼人, 大場 雄介, 河岡 義裕  日本女性科学者の会学術誌  20-  (1)  65  -66  2020/03
• 抗ラッサウイルス作用を有するリード阻害剤の創出

  水谷龍明, 浦田秀造, 水田賢志, 大場雄介  長崎大学熱帯医学研究拠点共同研究報告集  2018-  2019
• 【タンパク質・核酸の分子修飾】細胞質/オルガネラでの分子修飾 細胞内シグナル リン酸化(チロシン)

  笹島 仁, 大場 雄介  生体の科学  69-  (5)  428  -429  2018/10  [Not refereed][Not invited]
   

  ＜文献概要＞タンパク質のチロシン残基における可逆的なリン酸化は,細胞の増殖,分化,接着や免疫反応などにおけるシグナル伝達に関与する。チロシンリン酸化酵素自身におけるチロシンリン酸化は,酵素活性や細胞内局在,アダプタータンパク質との結合に変化をもたらし,基質のチロシンリン酸化を介してシグナル伝達を調節する。
• 少数性生物学ってなんだ?少数の要素を計測する―ウイルス何個で“感染”するか?

  藤岡容一朗, 田端和仁, 田端和仁, 大場雄介, 野地博行, 野地博行  実験医学  35-  (19)  3197‐3203  2017/12/01  [Not refereed][Not invited]
  
• RANKLによるインテグリンα2の発現亢進はインテグリンβ2の細胞内輸送を介して細胞接着を亢進する

  大場雄介, 山田珠希, 山田珠希, 山田珠希, 津田真寿美, 藤岡容一朗, 藤岡真理, 堀内浩水, 堀口美香, 佐藤絢, ネパール ブラバ, 王せい, 柏木彩花, 西出真也, 南保明日香, 芳賀永, 田中伸哉, 進藤正信  日本生理学雑誌(Web)  79-  (2)  49 (WEB ONLY)  -49  2017/05  [Not refereed][Not invited]
  
• インフルエンザウイルス細胞侵入において鍵となる宿主タンパク質の同定

  藤岡 容一朗, 西出 真也, 尾瀬 農之, 加藤 いづみ, 福原 秀雄, 藤岡 真理, 堀内 浩水, 佐藤 絢, Nepal Prabha, 柏木 彩花, Wang Jing, 堀口 美香, Paudel Sarad, 南保 明日香, 宮崎 忠昭, 前仲 勝実, 大場 雄介  日本細胞生物学会大会講演要旨集  69回-  61  -61  2017/05  [Not refereed][Not invited]
  
• インフルエンザウイルス細胞侵入において鍵となる宿主タンパク質の同定

  藤岡容一朗, 西出真也, 尾瀬農之, 加藤いづみ, 福原秀雄, 藤岡真理, 堀内浩水, 佐藤絢, NEPAL Prabha, 柏木彩花, WANG Jing, 堀口美香, PAUDEL Sarad, 南保明日香, 宮崎忠昭, 前仲勝実, 前仲勝実, 大場雄介  日本細胞生物学会大会(Web)  69th-  ROMBUNNO.T8‐04(P2‐028) (WEB ONLY)  -61  2017/05  [Not refereed][Not invited]
  
• 高速原子間力顕微鏡を用いた細胞表面膜構造動態のライブセルイメージング

  吉田藍子, 吉田藍子, 酒井信明, 植草良嗣, 粂田昌宏, 吉村成弘, 大場雄介  日本生化学会大会(Web)  90th-  2017
• Fluorescence bioimaging of intracellular signaling and its clinical application

  Yusuke Ohba, Yoichiro Fujioka  Journal of Oral Biosciences  58-  (4)  113  -119  2016/11/01  [Not refereed][Not invited]
   

  Background Fluorescent proteins have continued to shed light on cell biology since the cDNA of wild type green fluorescent protein was first isolated. Nowadays, these remarkable proteins are useful tools, not only in basic research, but also in clinical medicine. Highlight By taking advantage of fluorescent protein-based technologies, we identified a signaling network critical for influenza virus internalization and infection. In addition, we developed a highly sensitive biosensor for monitoring kinase activity that utilizes energy transfer between fluorescent proteins. This has led to a high-performance clinical test that enables the prediction of future therapeutic responses and the risk of acquired drug resistance for each individual patient before beginning molecular target therapy. Conclusion Technologies that utilize fluorescent proteins, such as the biosensor presented here, should find increasing applications in clinical medicine.
• CRKアダプター蛋白質はHGF/c-Metフィードバックループを介して膀胱癌のEMTと転移を誘導する

  王 磊, 松本 隆児, 津田 真寿美, 間石 奈湖, 安部 崇重, 木村 太一, 谷野 美智枝, 西原 広史, 樋田 京子, 大場 雄介, 篠原 信雄, 田中 伸哉  日本癌学会総会記事  74回-  J  -1142  2015/10  [Not refereed][Not invited]
  
• Tumor endothelial cells promote metastasis via biglycan secretion

  N. Maishi, Y. Ohba, K. Akiyama, N. Ohga, J. I. Hamada, M. T. Alam, M. Shindoh, Y. Hida, K. Hida  EUROPEAN JOURNAL OF CANCER  51-  S20  -S20  2015/09  [Not refereed][Not invited]
  
• Lysosomal interaction of Akt with Phafin2: A critical step in the induction of autophagy

  Masayuki Noguchi, Futoshi Suizu, Mami Lennikov-Matsuda, Bala Jyoti, Yusuke Ohba, Noriyuki Hirata  MOLECULAR CANCER THERAPEUTICS  14-  (7)  2015/07  [Not refereed][Not invited]
  
• IDENTIFICATION OF FIP1L1-PDGFRA ASSOCIATING MOLECULE THAT LOCATES IN THE NUCLEUS AND AUGMENTS THE ACTIVITY OF FIP1L1-PDGFRA

  T. Kondo, M. Ibata, J. Iwasaki, Y. Fujioka, K. Nakagawa, S. Darmanin, M. Onozawa, D. Hashimoto, Y. Ohba, S. Hatakeyama, T. Teshima  HAEMATOLOGICA  100-  327  -327  2015/06  [Not refereed][Not invited]
  
• MULTICENTER CLINICAL STUDY TO EVALUATE A UTILITY OF FRET-BASED DRUG SENRITIVITY TEST THAT PREDICTS EARLY MOLECULAR RESPONSE IN NEWLY DIAGNOSED CML PATIENTS TREATED WITH NILOTINIB

  T. Kondo, Y. Ohba, S. I. Fujisawa, T. Miyagishima, A. Mori, H. Iwasaki, M. Shindo, Y. Kakinoki, N. Kobayashi, S. Yamamoto, Y. Haseyama, S. Ando, N. Fujimoto, M. Nishio, M. Kurosawa, Y. Kohgo, T. Teshima  HAEMATOLOGICA  99-  340  -340  2014/06  [Not refereed][Not invited]
  
• インフルエンザウイルスのCa2+シグナルを介した宿主細胞侵入機構

  藤岡 容一朗, 津田 真寿美, 南保 明日香, 服部 ともえ, 佐々木 純子, 佐々木 雄彦, 宮崎 忠昭, 大場 雄介  日本細胞生物学会大会講演要旨集  66回-  103  -103  2014/05  [Not refereed][Not invited]
  
• インフルエンザウイルスのCa2+シグナルを介した宿主細胞侵入機構

  藤岡 容一朗, 津田 真寿美, 南保 明日香, 服部 ともえ, 佐々木 純子, 佐々木 雄彦, 宮崎 忠昭, 大場 雄介  日本細胞生物学会大会講演要旨集  66回-  146  -146  2014/05  [Not refereed][Not invited]
  
• 腫瘍血管内皮細胞のがん転移促進

  間石奈湖, 大場雄介, 大賀則孝, 大賀則孝, 秋山廣輔, 浜田淳一, 山本和幸, 川本泰輔, 進藤正信, 樋田泰浩, 樋田京子  日本病理学会会誌  103-  (1)  209  -209  2014/03  [Not refereed][Not invited]
  
• Ras-PI3Kシグナルが制御する外来因子取込み機構の解析

  藤岡 容一朗, 津田 真寿美, 服部 ともえ, 佐々木 純子, 佐々木 雄彦, 宮崎 忠昭, 大場 雄介  日本生理学雑誌  76-  (2)  81  -81  2014/03  [Not refereed][Not invited]
  
• 腫瘍血管内皮細胞のがん転移への関わり

  間石奈湖, 大場雄介, 樋田泰浩, 浜田淳一, 山本和幸, 大賀則孝, 秋山廣輔, 川本泰輔, 大澤崇宏, 近藤美弥子, 大村瞳, 井上農夫男, 進藤正信, 樋田京子  日本口腔科学会雑誌  63-  (1)  135  -135  2014/01  [Not refereed][Not invited]
  
• Ras-PI3Kシグナルが制御する外来因子取込み機構の解析

  藤岡 容一朗, 津田 真寿美, 服部 ともえ, 佐々木 純子, 佐々木 雄彦, 大場 雄介  日本細胞生物学会大会講演要旨集  65回-  153  -153  2013/05  [Not refereed][Not invited]
  
• 腫瘍血管内皮細胞のがん転移への関与

  間石奈湖, 大場雄介, 山本和幸, 大賀則孝, 浜田淳一, 秋山廣輔, 川本泰輔, 大澤崇宏, 大村瞳, 樋田泰浩, 進藤正信, 樋田京子  日本病理学会会誌  102-  (1)  437  -437  2013/04  [Not refereed][Not invited]
  
• SrcとAuroraキナーゼの2重阻害による滑膜肉腫の相乗的抗腫瘍効果

  新井 隆太, 渡部 琢哉, 三浪 明男, 津田 真寿美, 大場 雄介  北海道整形災害外科学会  54-  (2)  250  2013/03  [Not refereed][Not invited]
  
• がん転移における腫瘍血管内皮細胞の役割

  間石奈湖, 大場雄介, 大賀則孝, 秋山廣輔, 山本和幸, 浜田淳一, 川本泰輔, ALAM Mohammad T, 樋田泰浩, 樋田京子  日本血管生物医学会学術集会プログラム・抄録集  21st-  134  2013  [Not refereed][Not invited]
  
• がん微小環境における腫瘍血管の異常性獲得機構の解明

  樋田京子, 大賀則孝, 秋山廣輔, 間石奈湖, 川本泰輔, 大場雄介, 樋田泰浩  がん研究分野の特性等を踏まえた支援活動公開シンポジウムプログラム・抄録集 平成24年度  32  2013  [Not refereed][Not invited]
  
• がん転移における腫瘍血管内皮細胞の役割

  間石奈湖, 大場雄介, 大賀則孝, 秋山廣輔, 山本和幸, 浜田淳一, 川本泰輔, ALAM MOHAMMAD T, 大村瞳, 樋田泰浩, 樋田京子  日本がん転移学会学術集会・総会プログラム抄録集  22nd-  79  2013  [Not refereed][Not invited]
  
• SU6656はSrcとオーロラキナーゼの二重阻害によってin vivo滑膜肉腫に対する有効な抗腫瘍効果を示す

  新井 隆太, 渡部 琢哉, 津田 真寿美, 大場 雄介, 三浪 明男  日本整形外科学会雑誌  86-  (8)  S1101  2012/08  [Not refereed][Not invited]
  
• 抗ウイルス応答において活性化されるRIG-Iの強力な調節因子ZAPSの同定

  白鳥 聡一, 早川 清雄, 大和 弘明, 亀山 武志, 北辻 千展, 樫木 芙美, 後藤 翔平, 亀岡 章一郎, 藤倉 大輔, 山田 大翔, 水谷 龍明, 数馬田 美香, 佐藤 麻衣子, 田中 淳司, 浅香 正博, 大場 雄介, 宮崎 忠昭, 今村 雅寛, 高岡 晃教  北海道醫學雜誌 = Acta medica Hokkaidonensia  87-  (4)  194  -194  2012/08/01  [Not refereed][Not invited]
  
• RANKLはインテグリンα2の発現とエンドサイトーシスを介したインテグリンの細胞内輸送を亢進する

  我妻孝則, 津田真寿美, 山田珠希, 藤岡容一朗, 芳賀永, 戸塚靖則, 進藤正信, 大場雄介  日本分子生物学会年会プログラム・要旨集(Web)  35th-  3P-0363 (WEB ONLY)  2012  [Not refereed][Not invited]
  
• 腫瘍血管内皮細胞のがん転移への関わり

  間石奈湖, 大場雄介, 山本和幸, 大賀則孝, 浜田淳一, 秋山廣輔, 川本泰輔, 大澤崇宏, 近藤美弥子, 大村瞳, 樋田泰浩, 樋田京子  日本がん転移学会学術集会・総会プログラム抄録集  21st-  104  2012  [Not refereed][Not invited]
  
• DJ-1はシナプス小胞に局在する

  波田野 琢, 宇佐見 由希子, 久保 紳一郎, 大場 雄介, 有賀 寛芳, 斉木 臣二, 佐藤 栄人, 服部 信孝  Dementia Japan  25-  (3)  424  -424  2011/10  [Not refereed][Not invited]
  
• SrcおよびAuroraキナーゼ2重阻害による滑膜肉腫の相乗的in vivo抗腫瘍効果(Dual inhibition of Src and Aurora kinases abrogates tumor growth, invasion, and angiogenesis of synovial sarcoma in vivo)

  津田 真寿美, 新井 隆太, 渡部 琢哉, 尾瀬 農之, 小布施 力史, 前仲 勝実, 三浪 明男, 大場 雄介  日本癌学会総会記事  70回-  274  -274  2011/09  [Not refereed][Not invited]
  
• 慢性骨髄白血病に対する分子標的治療薬の反応性・抵抗性判定試験(Seeing response and resistance to BCR-ABL inhibition in chronic myeloid leukemia)

  大場 雄介, 金安 顕子, 水谷 龍明, ダルマニン・ステファニー, 近藤 健, 津田 真寿美  日本癌学会総会記事  70回-  242  -242  2011/09  [Not refereed][Not invited]
  
• RANKLは口腔癌細胞のインテグリンα2の発現と細胞接着を亢進する(RANKL upregulates integrin α2 expression and cell adhesion in oral cancer cells)

  大場 雄介, 山田 珠希, 藤岡 容一朗, 甲斐原 拓真, 戸塚 泰則, 進藤 正信, 津田 真寿美  日本細胞生物学会大会講演要旨集  63回-  156  -156  2011/05  [Not refereed][Not invited]
  
• SU6656 disturbs G2/M progression via inhibition of Aurora kinases

  Tsuda Masumi, Arai Ryuta, Ohba Yusuke  日本細胞生物学会大会要旨集  63rd-  159  2011/05  [Not refereed][Not invited]
  
• 染色体・核・遺伝子発現・シグナル伝達 インフルエンザウイルスは、カルシウムシグナル伝達によって、Ras-PI3K-仲介エンドサイトーシスを活性化する(Signal transduction/Chromosome/Cell nucleus/Gene expression Influenza viruses activate Ras-PI3K-mediated endocytosis via calcium signaling)

  藤岡 容一朗, 津田 真寿美, 服部 ともえ, 佐々木 純子, 佐々木 雄彦, 宮崎 忠昭, 大場 雄介  日本細胞生物学会大会講演要旨集  63回-  112  -112  2011/05  [Not refereed][Not invited]
  
• Analysis of interaction between tumor endothelial cells and tumor cells

  Nako Maishi, Noritaka Ohga, Yasuhiro Hida, Yusuke Ohba, Taisuke Kawamoto, Kosuke Akiyama, Kazuko Kitayama, Miyako Kondoh, Takahiro Osawa, Kazuyuki Yamamoto, Nobuo Inoue, Masanobu Shindoh, Kyoko Hida  CANCER RESEARCH  71-  2011/04  [Not refereed][Not invited]
  
• SU6656 suppresses human synovial sarcoma progression through Src and Aurora kinase inhibition

  Arai Ryuta, Tsuda Masumi, Watanabe Takuya, Ose Toyoyuki, Obuse Chikashi, Maenaka Katsumi, Minami Akio, Ohba Yusuke  日本分子生物学会年会プログラム・要旨集(Web)  34th-  WEB ONLY 2P-0690  2011  [Not refereed][Not invited]
  
• 自然免疫系における核酸認識受容体を介したシグナルの新規調節因子と抗ウイルス作用

  亀山武志, 早川清雄, 白鳥聡一, 大和弘明, 北辻千展, 樫木芙美, 後藤翔平, 亀岡章一郎, 藤倉大輔, 水谷龍明, 数馬田美香, 佐藤麻衣子, 今村雅寛, 浅香正博, 大場雄介, 宮崎忠昭, 高岡晃教  日本ウイルス学会学術集会プログラム・抄録集  58th-  220  2010/10/15  [Not refereed][Not invited]
  
• RANKL発現は腫瘍形成とEMTを亢進する(RANKL expression promotes tumorigenesis and epithelial mesenchymal transition)

  山田 珠希, 津田 真寿美, 戸塚 靖則, 進藤 正信, 大場 雄介  日本癌学会総会記事  69回-  494  -494  2010/08  [Not refereed][Not invited]
  
• Srcファミリーキナーゼ阻害薬はin vivo滑膜肉腫の増殖・浸潤・血管新生を抑制する

  新井 隆太, 渡部 琢哉, 津田 真寿美, 川口 秀明, 大場 雄介, 三浪 明男  日本整形外科学会雑誌  84-  (8)  S1244  2010/08  [Not refereed][Not invited]
  
• RANKLは微小環境特異的に発現し細胞接着・EMT・腫瘍形成を促進する生体内特異的機能マーカーである(RANKJL expression specifically observed in vivo promotes cell adhesion, epithelial mesenchymal transition, and tumor progression)

  山田 珠希, 津田 真寿美, 進藤 正信, 戸塚 泰則, 大場 雄介  日本細胞生物学会大会講演要旨集  62回-  196  -196  2010/05  [Not refereed][Not invited]
  
• メンブレントラフィック Ras-PI3Kシグナル経路がエンドサイトーシスとインフルエンザ感染を調節する(Membrane traffic The Ras-PI3K signaling pathway mediates endocytosis and the incorporation of influenza viruses)

  藤岡 容一朗, 津田 真寿美, 服部 ともえ, 佐々木 純子, 佐々木 雄彦, 宮崎 忠昭, 大場 雄介  日本細胞生物学会大会講演要旨集  62回-  117  -117  2010/05  [Not refereed][Not invited]
  
• 生体内腫瘍形成における腫瘍微小環境誘発性のRANKL発現の必要性(Requirement for tumor microenvironment-induced RANKL expression in the tumorigenesis in vivo)

  山田 珠希, 津田 真寿美, 戸塚 靖則, 進藤 正信, 大場 雄介  日本癌学会総会記事  68回-  47  -47  2009/08  [Not refereed][Not invited]
  
• FRETバイオセンサーを用いたCML分子標的薬剤の薬効評価系の構築(A FRET-based biosensor for the evaluation of the efficacy of molecular targeted drugs for chronic myeloid leukemia)

  水谷 龍明, 近藤 健, ダルマニン・ステファニー, 津田 真寿美, 田中 伸哉, 浅香 正博, 大場 雄介  日本癌学会総会記事  68回-  457  -457  2009/08  [Not refereed][Not invited]
  
• 悪性胸膜中皮腫におけるシグナル伝達アダプター分子CRKを介したRac活性の可視化

  谷野美智枝, 王磊, 津田真寿美, 西原広史, 大場雄介, 矢野聖二, 曽根三郎, 田中伸哉  日本呼吸器学会雑誌  47-  237  2009/05/10  [Not refereed][Not invited]
  
• Receptor activator of NF‐kB ligand (RANKL)発現口腔癌細胞株の悪性形質に関する機能解析

  山田珠希, 津田真寿美, 大場雄介, 川口秀明, 戸塚靖則, 進藤正信  日本病理学会会誌  98-  (1)  250  -250  2009/03/20  [Not refereed][Not invited]
  
• 慢性骨髄性白血病に対する分子標的治療薬の新たな薬効評価系の構築

  MIZUTANI TATSUAKI, KONDO TAKESHI, STEPHANIE DARMANIN, TSUDA MASUMI, KAWAGUCHI HIDEAKI, ASAKA MASAHIRO, OBA YUSUKE  日本病理学会会誌  98-  (1)  278  2009/03/20  [Not refereed][Not invited]
  
• Cell Motility and Invasion of Surviving Tumor Cells after 10 Gy Irradiation

  K. Tsutsumi, M. Tsuda, N. Yazawa, H. Nakamura, M. Yasuda, R. Yamazaki, H. Shirato, H. Kawaguchi, Y. Ohba, T. Nishioka  INTERNATIONAL JOURNAL OF RADIATION ONCOLOGY BIOLOGY PHYSICS  75-  (3)  S538  -S539  2009  [Not refereed][Not invited]
  
• Effect of Src family kinase inhibitor on proliferation and invasion in human synovial sarcoma in vivo

  Arai Ryuta, Tsuda Masumi, Watanabe Takuya, Kawaguchi Hideaki, Ohba Yusuke, Minami Akio  日本分子生物学会年会講演要旨集  32nd-  (Vol.1)  231  2009  [Not refereed][Not invited]
  
• Negative regulation of angiogenesis by zinc-finger transcription factor TCF8

  Takayuki Inuzuka, Masumi Tsuda, Shinya Tanaka, Yujiro Higashi, Hideaki Kawaguchi, Yusuke Ohba  JOURNAL OF MOLECULAR AND CELLULAR CARDIOLOGY  45-  S11  -S12  2008/10  [Not refereed][Not invited]
  
• 上皮細胞増殖因子(EGF)依存的なPTHrPの発現制御と口腔癌細胞の増殖能・局所浸潤能との関連性

  山田珠希, 津田真寿美, 大場雄介, 川口秀明, 戸塚靖則, 進藤正信  補体シンポジウム講演集  45th-  180  -180  2008/07  [Not refereed][Not invited]
  
• 口腔癌細胞における上皮細胞増殖因子受容体(EGFR)依存的なPTHrP発現制御と局所浸潤能との関連性

  山田珠希, 津田真寿美, 大場雄介, 戸塚靖則, 進藤正信, 川口秀明  適応医学  12-  (1)  25  -25  2008/05/20  [Not refereed][Not invited]
  
• Visualization of an interaction between Ras and Ras-binding domain in living cells by bimolecular fluorescence complementation

  Kaori Tsutsumi, Masumi Tsuda, Hideaki Kawaguchi, Yusuke Ohba  FASEB JOURNAL  22-  2008/04  [Not refereed][Not invited]
  
• 口腔癌細胞の接着に関連する分子の機能解析

  山田珠希, 津田真寿美, 大場雄介, 川口秀明, 戸塚靖則, 進藤正信  生化学  2P-1143  2008  [Not refereed][Not invited]
  
• 慢性骨髄性白血病に対する分子標的治療薬の新たな薬効評価系の構築

  MIZUTANI TATSUAKI, KONDO TAKESHI, DARMANIN STEPHANIE, TSUDA MASUMI, KAIHARA TAKUMA, KAWAGUCHI HIDEAKI, ASAKA MASAHIRO, OBA YUSUKE  生化学  4P-1171  2008  [Not refereed][Not invited]
  
• ParkinによるPINK1の安定化機構(平成18年度老人性疾患病態・治療研究センター研究発表会)

  柴 佳保里, 新井 健夫, 久保 紳一郎, 大場 雄介, 水野 美〓, 服部 信孝  順天堂医学  53-  (3)  521  -522  2007/09
• EGFシグナル伝達はPTHrPの発現を誘導し、口腔癌細胞の悪性化に関与する(EGF signaling regulates PTHrP expression and malignancies in oral cancer cells)

  山田 珠希, 津田 真寿美, 大場 雄介, 戸塚 靖則, 進藤 正信  日本癌学会総会記事  66回-  501  -501  2007/08  [Not refereed][Not invited]
  
• 植物におけるsmall RNA生成/制御の場の解析

  藤岡容一朗, 内海真希, 岩崎信太郎, 大場雄介, 渡辺雄一郎  日本RNA学会年会要旨集  9th-  138  2007/07/28  [Not refereed][Not invited]
  
• 慢性骨髄性白血病に対する新たな診断技術の開発

  MIZUTANI TATSUAKI, TSUDA MASUMI, KAWAGUCHI HIDEAKI, OBA YUSUKE  生化学  4P-0935  2007  [Not refereed][Not invited]
  
• (345) Formulation of in planta FRET System for Analysis of Viral Movement(Abstract of the Paper Presented at the 2006 Annual Meeting in Sapporo)

  Fujioka Y., Ohba Y., Watanabe Y.  Annals of the Phytopathological Society of Japan  72-  (4)  294  -294  2006/11/25
• 免疫2006 7章 病気と免疫 病原体認識受容体シグナルにおけるIRFファミリー転写因子の役割

  高岡晃教, 柳井秀元, 本田賢也, 大場雄介, 谷口維紹  Mol Med  42-  308  -317  2005/12/15  [Not refereed][Not invited]
  
• MyD88‐IRF7シグナル伝達系の時空間制御によるIFN産生機構

  大場雄介, 本田賢也, 柳井秀元, 高岡晃教, 谷口維紹  日本癌学会学術総会記事  64th-  523  2005/08/15  [Not refereed][Not invited]
  
• I型インターフェロン依存性免疫応答における主要な調節因子としてのIRF‐7の役割

  柳井秀元, 本田賢也, 大場雄介, 高岡晃教, 吉田進昭, 谷口維紹  日本癌学会学術総会記事  64th-  523  2005/08/15  [Not refereed][Not invited]
  
• 樹状細胞群の分化と活性化の分子機構

  HONDA KEN'YA, YANAI HIROYUKI, MIZUTANI TATSUAKI, NEGISHI HIDEO, OBA YUSUKE, TAKAOKA AKINORI, TANIGUCHI TADATSUGU  日本分子生物学会年会プログラム・講演要旨集  27th-  345  2004/11/25  [Not refereed][Not invited]
  
• 樹状細胞群におけるIRFファミリー転写因子の役割

  YANAI HIDEYUKI, HONDA KEN'YA, ASAGIRI MASATAKA, NEGISHI HIDEO, MIZUTANI TATSUAKI, SHIMADA NAOYA, YOSHIDA NOBUAKI, OBA YUSUKE, TAKAOKA AKINORI  日本分子生物学会年会プログラム・講演要旨集  27th-  593  2004/11/25  [Not refereed][Not invited]
  
• RalA activation at nascent lamellipodia of EGF-stimulated Cos7 cells and migrating MDCK cells

  Akiyuki Takaya, Yusuke Ohba, Kazuo Kurokawa, Michiyuki Matsuda  CELL STRUCTURE AND FUNCTION  29-  57  -57  2004/05  [Not refereed][Not invited]
  
• Regulation of a Small GTPase Rap2

  OHBA Yusuke  北海道醫學雜誌 = Acta medica Hokkaidonensia  76-  (1)  13  -20  2001/01/01
• Activation of the ERK/MAPK pathway by an isoform of Rap1gap associated with G alpha I

  N Mochizuki, Y Ohba, K Nagashima, A Endo, M Matsuda  CIRCULATION  102-  (18)  353  -353  2000/10  [Not refereed][Not invited]
  

Books etc

• Oral Cancer

  IN TECH 2012
• Bio Science 新用語ライブラリー免疫

  羊土社 2000

Association Memberships

• PHYSIOLOGICAL SOCIETY OF JAPAN   日本細胞生物学会   日本生化学会   日本癌学会   日本分子生物学会   

Research Projects

• Visualization of symbiotic interfaces and cellular responses

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

  Date (from‐to) : 2020/11 -2025/03 

  Author : 大場 雄介

   

  研究で実施する、細胞表面で生じるマテリアルと細胞表面タンパク質の「弱い相互作用」が生じている現場である細胞膜の動態観察を実現するため、高速ライブセル原子間力顕微鏡（atomic force microscopy，AFM；以下、高速ライブセルAFM）および蛍光顕微鏡による相関イメージング（correlative light and acomic force microscopy, CLAM）を実装した。また、領域内の測定拠点としての活動が円滑に行えるように、体制整備および予備実験が完了した。これによりマテリアルー生体の「弱い相互作用」の現場の「まるごと」観察を実現した。 【AFM】ウイルスや種々の外来因子を用いて、柔らかい細胞膜上の6.0 μm × 4.5 μm ×1.0 μmの範囲において、xyzすべての方向に対して10 nmの等方性空間分解能、また0.5フレーム/秒（2秒に1枚の撮像）以上の時間分解能を両立した観察系を複数セットアップした。これらの装置の性能について確認し、予定通りのスペックが出 ていることを確認している。 【蛍光顕微鏡】分子同定や細胞応答の可視化に欠かせない蛍光イメージングについては、その分解能を向上するために超解像技術を導入した。また、薄層斜光証明法（highly inclined and laminated optical sheet microscopy，HILO）により細胞膜頂端膜の局所励起と一分子イメージングが可能な照明・観察光学系を、AFMが実装済みの顕微鏡にインストールした。 さらに、「弱い相互作用」をCLAM上でどのように可視化するかについて予備的検討を、ウイルス粒子と細胞膜との関係をモデルに行った。その結果、相互作用がない場合には粒子は細胞膜上にとどまることができないこと、相互作用の強弱により側方拡散係数（lateral diffusion coefficient）が異なることを新たに見出した。これによりマテリアル一つの解像度で「弱い相互作用」を定量解析する準備が整った。
• Biophysical Chemistry for Material Symbiosis

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

  Date (from‐to) : 2020/11 -2025/03 

  Author : 山吉 麻子, 前仲 勝実, 荏原 充宏, 望月 慎一, 長谷 耕二, 大場 雄介, 宇都 甲一郎, 白石 貢一, 植畑 拓也, 森 健, 天野 麻穂, 山本 剛史

   

  本研究課題の目的は、学術変革領域「マテリアル・シンバイオシスのための生命物理化学」の研究推進と領域運営である。 【領域会議等の開催】2021年度9月より公募研究班の領域参画が始まった。領域目標を達成するためには、個々の研究の活性化に加え、領域全体が一体となって相互補完的な融合研究を実施することが肝要である。そこで、領域全メンバー参加型の第１回領域会議（2021/11/4-11/5）をハイブリッド開催し、最新の研究成果の共有と共同研究推進の場を提供した。また別途、A01-A03各班での班会議を開催し情報共有を行った。総括班員による班会議も定期開催し、領域体制の最適化に勤めた。 【領域共催のシンポジウムの開催】日本薬剤学会第36年会において、ラウンドテーブル「シンバイオティック・マテリアルの実現と新しい創薬モダリティを考える」を企画・開催した（2021/1/13）。また、日本DDS学会第37年会において、若手ワークショップ「腸内細菌叢の共生原理に学ぶDDS」を企画・開催した（2019/6/29）。領域メンバーはもとより、国内外研究協力者が領域研究に関する研究発表を行い、領域研究を外部に向けて発信した。さらに、男女共同参画推進を目的としたシンポジウムを共催した（「北九州サイエンスガールプロジェクト（北九州市立大学公開講座）」2021/10/2, 10/9, 10/16; 「令和3年度 長崎大学リケジョ育成プログラム 志セミナー」, 2021/12/4）。 【イメージングブートキャンプ】領域班員、若手研究者に、物質共生学研究で重要となる細胞イメージング技術を修得する場を提供することを目的に、イメージングブートキャンプ2021 （2021/9/7-9）を共催した。 【共同研究推進】分野横断的かつ積極的な共同研究を推進するために、総括班よりマテリアル合成支援や超高速AFM解析支援等を提供した。
• 一次線毛とコロナウイルス感染におけるACE2の役割の解明

  日本学術振興会：科学研究費助成事業

  Date (from‐to) : 2021/11 -2024/03 

  Author : 大場 雄介, PANINA YULIA

   

  本研究は、SARS-CoV-2受容体ACE2について、基礎的な細胞プロセスやCOVID-19症状の重症度との関連を理解すること目的に、特に一次繊毛との関連性に着目して研究を進めている。最終的には、ACE2が繊毛に局在する分子メカニズムの解明、SARS-CoV-2とACE2の結合が繊毛に与える影響、高血圧・糖尿病病態がこれらにどのような影響を与えるのかの解明を目指す。 本年度、つまり研究開始から6ヶ月間の到達目標は、ACE2の細胞内局在を決定し、ACE2発現細胞と非発現細胞との違いを調べることであった。まず、ACE2の局在を研究するのに適したモデル細胞を決定した。いくつかの細胞株をテストした結果、ヒトBEAS-2B細胞株、マウス退治線維芽細胞株、マウス3T3細胞株が、繊毛の識別性およびACE2抗体に交差性から適したモデルであると判断した。次に、市販のACE2抗体を複数用いて免疫蛍光法を行ったところ、ACE2を検出できるのは一つのみであることが判明した。この抗体を用いて、ACE2が細胞膜に局在していることを確認した。また、ACE2を恒常的に過剰発現させたBEAS-2B細胞株を用い、元のACE2非発現BEAS-2B細胞株と比較したところ、細胞の構造、形状、細胞骨格に大きな変化があることを見いだした。さらに、ACE2の過剰により細胞形態が変化することが確認されたため、細胞接着を担う細胞膜タンパク質を探索し、その局在が変化する接着分子を同定した。一方、生きた細胞での局在を、蛍光タンパク質タグしたACE2で検討しようと試みたが、繊毛局在性は認められなかった。
• オルガネラバイオロジーの新展開

  日本学術振興会：科学研究費助成事業

  Date (from‐to) : 2021/07 -2023/03 

  Author : 大場 雄介

   

  我々は最近、EGF刺激によりエンドサイトーシスを誘導した時、細胞膜から小胞が形成される初期段階で、初期エンドソームマーカーであるRab5と後期エンドソームマーカーであるRab7が長時間同時に局在する小胞が存在することを見出した。このような遷移状態のオルガネラはこれまでに報告がなく、その形成メカニズムや役割は未知である。そこで、この小胞の特徴や運ばれる物質、形成に関わる因子を調べ、Rab5/7ダブルポジティブ小胞の生理的意義を明らかにすることを目指した。はじめに、Rab5/7ダブルポジティブ小胞に局在する分子を調べ、EEA1およびSNX5が共局在することがわかった。Rab5/7ダブルポジティブ小胞は2-5 μmの大きい小胞であり、また、この小胞と共局在するSNX5はマクロピノソームのマーカー分子として知られていることから、Rab5/7ダブルポジティブ小胞はマクロピノサイトーシスによって形成されるマクロピノソームであることが示唆された。 次に、この小胞によって輸送される物質および輸送経路を調べた。Rab5/7ダブルポジティブ小胞はEGF刺激後に形成されることから、EGF受容体がこの小胞内に含まれているかどうかを調べた。その結果、Rab5/7ダブルポジティブ小胞とRhodamine標識EGFは共局在しており、この小胞はEGF受容体を輸送していることが示唆された。また、Rab5/7ダブルポジティブ小胞の挙動をタイムラプスイメージングで観察したところ、リソソームマーカーのみと共局在したことから、この小胞は分解経路に輸送されることが示唆された。
• エンドソームとミトコンドリアの連携ゾーンによる細胞生理機能制御機構の解明

  日本学術振興会：科学研究費助成事業

  Date (from‐to) : 2020/04 -2022/03 

  Author : 大場 雄介

   

  Ras-PI3Kシグナルの制御ペプチドRAPEL配列の結合因子探索から同定したミトコンドリア外膜タンパク質voltage-dependent anion chanle 2（VDAC2）の機能解析を行った。これまでに、VDAC2は生化学的にRAPELと結合すること、ノックダウンによりRas-PI3Kのエンドソーム局在、クラスリン非依存性エンドサイトーシス、およびエンドソーム成熟化が抑制されること、過剰発現によりこれらが亢進することを確認した。すなわち、VDAC2はRas-PI3Kによるエンドサイトーシスの制御を正にコントロールする因子であることが明らかになった。 ミトコンドリア外膜タンパク質であるVDAC2が、どのようにしてエンドサイトーシス制御に関与するかを明らかにするため、我々が開発したオルガネラマーカーを用いて、生細胞タイムラプスイメージングを行った。それにより、ミトコンドリアとエンドソームの間には、一過性に繰り返す相互作用が生じることを発見した。この相互作用は、VDAC2のノックダウンで減少した。さら、ミトコンドリアと接触するエンドソーム上では、早期エンドソームマーカーの蛍光強度が減少し、接触過程でエンドソームが成熟化することが示唆された。実際、ミトコンドリアとの接触中にpHが低下することがわかった。さらに、光遺伝学手法を用いてミトコンドリアとエンドソームの接触を誘発したところ、エンドソームの成熟化が進み、VDAC2ノックダウンでその成熟化が抑制された。したがってVDAC2はエンドソーム とミトコンドリアのtethering factorであると同時に、その連携ゾーンにおける働きが重要であることが示唆された。野生型のVDAC2発現はVDACノックダウンを補完できるが、ある変異型VDAC2はそれが不可能であることから、連携ゾーンでのVDAC2の機能解明を目指す。
• 蛍光偏光を用いた等方性分解能顕微鏡の開発と細胞膜動態の可視化

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Fund for the Promotion of Joint International Research (Fostering Joint International Research (B))

  Date (from‐to) : 2019/10 -2022/03 

  Author : 大場 雄介, 藤岡 容一朗, 佐藤 絢
• 細胞膜動態の高分解能計測によるエンドサイトーシス超初期過程の分子基盤の解明

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Challenging Research (Exploratory)

  Date (from‐to) : 2019/06 -2021/03 

  Author : 大場 雄介
• インフルエンザウイルス感染におけるシンギュラリティ

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research on Innovative Areas (Research in a proposed research area)

  Date (from‐to) : 2019/04 -2021/03 

  Author : 大場 雄介
• Analysis of the communication zone between endosomes and mitochondria

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research on Innovative Areas (Research in a proposed research area)

  Date (from‐to) : 2018/04 -2020/03 

  Author : 大場 雄介

   

  本研究は、低分子量GTP結合タンパク質Rasと、その標的因子の1つであるphosphoinositide 3-kinase (PI3K)が、他の標的因子とは異なり上皮増殖因子 (epidermal growth factor, EGF) 刺激依存的にエンドソームでRasと複合体を形成する分子メカニズムを解明することから開始した。 Rasの主な標的因子のRas-binding domain（RBD）のアミノ酸配列を比較にり、PI3K特異的配列を見出し、Ras-PI3K endosomal localization（RAPEL）配列と命名した。RAPEL結合因子を酵母ツーハイブリッド法で探索したところ、ミトコンドリア外膜タンパク質voltage-dependent anion channel 2（VDAC2）が同定された。VDAC2のノックダウンによりRas-PI3K複合体のエンドソーム局在、クラスリン非依存性エンドサイトーシス、エンドソームの成熟化が抑制された。 次に、ミトコンドリアタンパク質がいかにしてエンドサイトーシスの制御に関与するかを調べるため、生細胞内でミトコンドリアとエンドソームのダイナミクスを詳細に観察したところ、両者は一過性に近接していることが明らかになった。この近接は、コントロール細胞ではEGF刺激後で促進されたのに対し、VDAC2ノックダウン細胞では変化が認められなかった。さらに光遺伝学を用いてミトコンドリア―エンドソーム間相互作用を青色光により誘導する系を構築したところ、相互作用依存性にエンドソームの酸性化が促進された。したがって、エンドソームとミトコンドリア間の接触ゾーンの形成がエンドソームとの成熟化を促進すること、RAPELとVDAC2がゾーン形成のtetheringに関与することが示唆された。
• エンドソームとミトコンドリアの物理的相互作用と機能連関による細胞機能の調節機構

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)

  Date (from‐to) : 2017/04 -2020/03 

  Author : 大場 雄介

   

  昨年度に引き続き、低分子量Gタンパク質Rasと標的分子複合体のうち、Ras-PI3K複合体のみがエンドソーム移行する分子メカニズムの解明を目指し研究を行った。標的因子のRas-binding domain（RBD）のアミノ酸配列解析により、PI3K-RBDに存在する特徴的な配列を同定し、この配列を欠損させるとRas-PI3Kのエンドソーム局在が抑制されたことから、Ras-PI3K endosomal localization domain（RAPEL domain）と命名した。RAPEL過剰発現によりエンドサイトーシスが抑制され、エンドサイトーシスを介して細胞に侵入するインフルエンザウイルスの感染も抑制された。さらに、RAPELと細胞膜透過性ペプチド（cell penetrating peptide, CPP）との誘導ペプチドを合成した。ペプチドを導入した細胞にインフルエンザウイルスを暴露したところ、感染抑制効果が認められた。さらに、ペプチドによる感染抑制効果はヒト呼吸上皮細胞を用いたin situでも認められた。以上より、RAPELはペプチド療法にも応用可能であることが示された。これらの結果は、学術雑誌に受理された。 一方、上記の結果は、RAPELに結合する因子がRas-PI3K複合体のエンドソーム移行とエンドサイトーシスを制御することを示唆するものである。質量分析法と酵母ツーハイブリッド法によるスクリーニングで得られたRAPEL結合因子計50個のうち、6因子についての解析が進んでいる。うち3因子の解析が終了し論文を作成・投稿準備中である。因子Aについては新規のオルガネラ間相互作用を担うタンパク質であることが判明した。因子Bはイノシトールリン脂質依存的にRas-PI3Kのエンドソーム局在の制御因子であることを突き止めた。因子Cについては上記二つの負の制御因子であった。
• Configuration of cell axes for the development of cell authentication system

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Challenging Research (Exploratory)

  Date (from‐to) : 2017/06 -2019/03 

  Author : Ohba Yusuke

   

  In order to set a "template" for authenticating cells and a "coordinate axis" for describing it, parameters regarding the arrangement of each organelle were obtained. For this purpose, an organelle marker library was constructed using various fluorescent proteins. It was found that the facilitation of the folding of fluorescent proteins might negatively affect the localization of the organelle markers. In addition, by analyzing the arrangement of organelles using the library, it was found that the centroids of the endosomal system formed a certain line in cells.
• 細胞競合現象の時空間的解析とその操作

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research on Innovative Areas (Research in a proposed research area)

  Date (from‐to) : 2017/04 -2019/03 

  Author : 大場 雄介

   

  本研究は、光による細胞競合の誘導系を開発し、細胞競合の発生を時空間的に厳密に制御すること、同時に分子の活性化、分子間相互作用、イオン濃度変化等を蛍光バイオイメージングで観察し、winner、looser間での細胞競合が生じるための時空間的な分子動態を詳細に明らかにすることを目的に行っている。 昨年度までに、488 nmの光で分子間相互作用が誘導されるCry2-CIBの系を用いて、二つのRas活性化法（RasV12とSosを用いるもの）を構築した。この二つの系を用いて、RasV12の系のみで細胞競合が観察され、Sosによる内在性Rasの活性化では細胞競合が誘導されないことを見出した。この結果は、細胞競合において何がwinnerとlooserを決めるかという根本的課題に迫る緒になると期待された。 しかし、この系では青色光の刺激が必要なため、シグナル伝達研究に有効なフェルスター共鳴エネルギー移動（Foerster resonance energy transfer, FRET）や蛍光タンパク質再構成法（bimolecular fluorescence complementation, BiFC）等の青色光を励起光として用いるイメージング手法との併用が難しい。そこで、それらイメージング手法との併用を目指し、遠赤色光受容タンパク質phytochromeB（PhyB）とphytochrome interaction factor 3（PIF3）の結合を遠赤色光により誘導できるオプトジェネティクスの手法を取り入れた。これにより、遠赤色光照射によりSrcを細胞質から細胞膜あるいはエンドソームに移行させることに成功した。また、様々なオルガネラに局在するPhyBとPIF3を作製し、異種オルガネラ間相互作用を誘導する系も併せて開発した。
• Drug screening to improve mitochondrial functions

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

  Date (from‐to) : 2016/04 -2018/03 

  Author : Imai Yuzuru, SHIBA Kahori, INOSHITA Tsuyoshi, OHBA Yusuke, ARANO Taku

   

  The genes responsible for autosomal recessive early-onset Parkinson’s disease, Parkin and PINK1 encode a ubiquitin ligase and a mitochondrial protein kinase, respectively. A series of studies have revealed that Parkin in collaboration with PINK1 has a role for mitochondrial quality control, impairment of which leads to dopaminergic neurodegeneration. In this study, we aimed at searching drugs to improve mitochondrial functions through moderate activation of PINK1-Parkin signaling. We developed a cell-based reporter assay to monitor PINK1-Parkin signaling and screened approximately 310-thousands of chemicals. Hit compounds obtained in the screening were further tested for mitochondrial membrane potential and cell viability and three candidates were identified as seed compounds.
• Intracellular signaling machinery that regulates endocytosis and uptake of exogenous factors(Fostering Joint International Research)

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Fund for the Promotion of Joint International Research (Fostering Joint International Research)

  Date (from‐to) : 2016 -2018 

  Author : Ohba Yusuke, Tani Tomomi

   

  We have developed a technology to obtain information on "angle" in biological phenomena by measuring the "orientation" of molecules using the properties of polarization and anisotropy of fluorescence. In particular, we established a method to detect the changes in the angle (curvature) of cell membranes by translating it to changes in fluorescence intensity corresponding to alteration of the molecular orientation. With these advantages, the curvature of the plasma membrane and its change, which had been visible only observable with electron microscopy, can be optically observed at a high time resolution (second level), making possible to visualize cell membrane deformation in the very early stage of endocytosis.
• ウイルス感染コンピテンシーの不均一性を決定する宿主因子の同定

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research on Innovative Areas (Research in a proposed research area)

  Date (from‐to) : 2015/04 -2017/03 

  Author : 大場 雄介

   

  本研究では、インフルエンザウイルスの感染コンピテンシーを規定する因子の同定をその機能解析を行うことを目的に研究を進めている。これまで我々は、インフルエンザウイルスの細胞内取込において、カルシウムシグナルが重要であることを報告しており、本研究では細胞膜に存在するカルシウム制御に関与する分子に着目して研究を行っている。昨年度までに、ある細胞膜タンパク質がその制御に重要であることを見出した。この因子のノックダウンおよび薬剤での阻害によって、インフルエンザウイルス感染時のカルシウム応答は完全に抑制され、またプラークアッセイを用いた実験においてもインフルエンザ感染を強く抑制した。また、この分子の発現量とウイルス感染効率に強い相関があったことから、当該因子がウイルスが宿主細胞への感染過程において重要な役割と担っているおり、ウイルス感染コンピテンシーの規定因子の1つであるという観点から研究を進めた。 この因子はウイルス粒子のHAタンパク質と結合することが昨年までに明らかとなっていたため、当該因子のC末端細胞表面部位に存在するシアル酸化の標的になるアスパラギンをグルタミンに置換した変異体を作製した。変異体とHAの結合は野生型のそれと比べて減少していた。また、当該因子をノックダウンした細胞に野生型を戻すと、細胞内カルシウムの上昇とインフルエンザウイルス粒子の取り込み、インフルエンザ感染の全てが回復したが、変異型の発現を戻しても回復効果は認められなかった。以上から、シアル酸を介した当該因子とウイルス粒子の結合が、ウイルス感染時のカルシウム動員と粒子の取り込み、その後の感染成立に重要であることが示された。本研究成果は学術雑誌に投稿中である。
• Investigation of a physiological role of endosome-mitochondrial interaction by fluorescence bioimaging

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

  Date (from‐to) : 2015/04 -2017/03 

  Author : Ohba Yusuke, Nanbo Asuka, Nishide Shinya, Fujioka Yoichiro, Fujioka Mari, Horiuchi Kosui, Satoh Aya O.

   

  We have reported that the complex of small G protein Ras and its target molecule PI3K is localized in the endosomes and controls endocytosis and viral particle internalization. However, the molecular mechanism by which the Ras-PI3K complex is localized is the endosome remains unknown. In this study, the amino acid sequence responsible for the above phenomenon was identified. By screening the binding protein for the sequence, a mitochondrial protein was identified. Knockdown of this mitochondrial protein inhibited the translocation of Ras-PI3K complex to the endosome, endocytosis, and mitochondrial-endosome interaction, suggesting that binding of PI3K and the factor regulates endocytosis through interaction between organelles.
• Intracellular signaling machinery controlling endocytosis and uptake of extracellular materials

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)

  Date (from‐to) : 2014/04 -2017/03 

  Author : Ohba Yusuke, Nanbo Asuka, Nishide Shinya, Fujioka Yoichiro, Fujioka Mari, Horiuchi Kosui, Satoh Aya O.

   

  Endosome is a multifunctional platform through the emission and the regulation of a variety of signal transduction pathways. However, the relationship between signaling dynamics and cell function via the endosomes in living cells has yet to be analyzed in detail. In this study, we demonstrated that angiotensin II (AII) type 2 receptor (AT2R) negatively regulates type 1 receptor (AT1R)-signaling through a series of events including heterodimerization of AT1R and AT2R, internalization via endocytosis of the complex, change in the molecular orientation of receptors in the complex. We also clarified that activation of both receptors and and phosphorylation of serine residues at the C-terminus of AT2R by protein kinase C (PKC) are indispensable for this function.
• バイオイメージングによるウイルス感染と細胞応答の定量解析

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research on Innovative Areas (Research in a proposed research area)

  Date (from‐to) : 2014/04 -2016/03 

  Author : 大場 雄介

   

  本研究においては、細胞への感染成立には、インフルエンザウイルス粒子が何個必要かという命題を解くために、研究を行っている。そのため、ウイルス粒子が細胞内侵入する際に重要なシグナル伝達経路の解析と、ウイルス粒子の精密計測により粒子数とその経路の関係性を検討した。シグナル伝達解析においては感染時のカルシウム上昇に着目し、その責任分子の機能解析を進めた。同定した責任分子は24回膜貫通型の膜タンパク質であり、生化学的解析を行うための可溶化が困難であった。種々の界面活性剤を用いて可溶化条件を検討したところ、凝集せずに単量体として可溶化することに成功した。この条件を用いることで、この責任分子とウイルス粒子の結合を介してカルシウム上昇が引き起こされることが明らかになった。 また、領域内の東京大学工学系研究科・野地先生、田端先生との共同研究により、用いる ウイルスサンプルの粒子数とMOIの関係を明らかにしたうえで、感染性の評価を行った。その結果、ウイルス感染効率はこれまで考えられていたMOIよりも、粒子数に依存することが明らかになった。また、粒子数が多い時と少ない時では異なる細胞の応答性が観察された。粒子数が多い時には感染にある種の正のフィードバックのようなブースト機構が存在すること、そのブースト機構にカルシウムが重要であることが解った。実際に高い複製が認められ、多くの粒子が取り込まれたと考えられる細胞には、上記の責任分子が多く発言していた。一方で、複製が盛んではない（0ではない）細胞においては上記の責任分子の発現量が低く、カルシウム応答も見られないことから、カルシウム非依存性のメカニズムによりウイルスが感染するものと考えられた。両者の境界は細胞1つあたりウイルス粒子20個程度であり、感染には数10個程度の粒子が必要であることが明らかになった。
• Study on EGFR-TKI resistance using FRET biosensor

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

  Date (from‐to) : 2014/04 -2016/03 

  Author : Akita Hirotoshi, Ohba Yusuke, Kinoshita Ichiro

   

  EGFR-TKI resistant cells were isolated in three non-small cell lung cancer (NSCLC) cell lines, using FRET biosensor and flow cytometer. cDNA microarray analysis showed common gene expression changes among the resistant cells. Annotation analysis revealed three up-regulated pathways and five down-regulated pathways. The ABC transporter system was included in the genes and pathways, which were found to be changed in relation to the acquired resistance, suggesting that this system could be involved in the resistance to EGFR-TKI in NSCLCs.
• Analyzing the mechanism of TKI-resistance in Ph-positive ALL

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)

  Date (from‐to) : 2013/04 -2016/03 

  Author : KONDO TAKESHI, OHBA Yusuke, MINAMI YOSUKE

   

  Using the FRET-based drug sensitivity test, we identified and isolated TKI-rafractory cell population in Ph-positive ALL. Analysis of gene expression profile in TKI-refractory cells indicated that so many intracellular pathways were activated. One of them was sumoylation pathway. In vitro analysis, we proved that BCR-ABL is sumoylated. Moreover, inhibition of sumoylation effectively induced apoptosis in Ph+ALL cells.
• Multidimendional FRET imaging of intracellular signal transduction

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research on Innovative Areas (Research in a proposed research area)

  Date (from‐to) : 2010/04 -2016/03 

  Author : Matsuda Michiyuki, KIYOKAWA Etsuko, HIRATSUKA Takuya, OHBA Yusuke, AOKI Kazuhiro, NAKAMURA Takeshi

   

  FRET biosensors visualize activities of signaling molecules in living cells. However, the use was limited mostly to the cultured 2D cells. Here, we aimed at developing technologies to apply FRET biosensors in 3D culture cells and in living tissues. Due to high recombination rates, stable expression of FRET biosensors had been a difficult task. To overcome this problem, we employed the transposon-mediated gene transfer. We found either piggcBac transposase or Tol2 trasnpoase worked efficiently for the gene transfer of FRET biosensor cDNAs. In particular, we found tol2-mediated gene transfer worked efficiently to develop transgenic mice highly-expressing FRET biosensors, which we named FRET mice. By using FRET mice, we observed activation of various signaling molecules in living tissues.
• Challenge to the accurate detection of minimal drug resistant cells

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

  Date (from‐to) : 2013/04 -2015/03 

  Author : OHBA Yusuke

   

  In Philadelphia chromosome-positive leukemia, growth and survival of leukemia cells solely depend on constitutive tyrosine kinase activity of new fusion gene product BCR-ABL formed by chromosome reciprocal translocation between chromosomes 9 and 22. Therefore, the treatment for this disease has been radically improved by the introduction of the specific tyrosine kinase inhibitor of BCR-ABL. However, it is desired to develop new technologies that enable the detection of a minimal number of drug-resistant cells because substantial patients experience drug resistance regardless of the presence or absence of past and current treatment. We have previously developed a novel technique to detect relatively small number of drug resistant cells. Here, we challenged to centuplicate the detection ability by the improvement of the FRET biosensor, the detection method and the analytical method. As a result, we obtained 162-fold improvement throughout this study.
• Analysis of signal transduction pathways evoked by virus infection

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)

  Date (from‐to) : 2011/04 -2014/03 

  Author : OHBA Yusuke, TSUDA Masumi

   

  Virus infections are one of the largest threats of humanity in modern society with scientific and technological advances, of which the H1N1 pandemic in 2009 would be a practical example. We have previously reported that PI3K bound to and activated by Ras plays a crucial role in the regulation of endocytosis by deciphering the signal transduction pathways with the best use of fluorescence bioimaging techniques. In this study, we found that Ras-PI3K signaling is required for influenza virus infection, and identified intracellular calcium as a key molecule to regulate a series of influenza virus infection processes. It might also be expected to lead to a new concept of antivirus therapeutics through application of these results.
• 微小環境の定量解析に資する培養・イメージングシステムの構築

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research on Innovative Areas (Research in a proposed research area)

  Date (from‐to) : 2011/04 -2013/03 

  Author : 大場 雄介

   

  本研究では、腫瘍微小環境を舞台にがん細胞と間質細胞等が演じるドラマを分子レベルで解明する実験系の提供を第一目的とし、昨年度から研究を実施している。大きく分けて以下の2つの研究項目に取り組み、複雑系システムを理解するために多次元イメージング系の基礎を確立した。 細胞の蛍光ラベル：様々な蛍光タンパク質により細胞内のオルガネラをマークする発現ベクターを構築した。蛍光タンパク質については、Sirius、CFP、YFP、tdTomato、TurboFP650、KeimaおよびEGFPの7色それぞれについて、核(ヒストン)、細胞膜 、細胞膜、アクチン細胞骨格、細胞接着斑、早期/後期エンドソーム、ミトコンドリア、微小管、小胞体、ゴルジ装置、リサイクリングエンドゾームのオルガネラ局在発現ベクターを完成させた （計84種）。 3次元培養系の確立：前項で樹立した多彩な蛍光タンパク質でラベルした細胞を用いて、腫瘍微小環境を模倣した3次元培養系を構築した。 この培養条件下において液性因子や基質種類・混合比を調整しながら細胞の挙動を顕微鏡観察し、そのダイナミクスを時空間的に解析した。実際、扁平上皮癌細胞を用いた癌組織の形成を試験管内で再構成することに成功するとともに、その過程においての細胞の挙動の観察にも成功した。また、以前我々が同定した扁平上皮癌細胞の悪性度制御因子をこの系に用いることにより、高分化型扁平上皮癌から低分化型の癌組織を誘導することにも成功した。このことは、本研究で確立した3次元培養系が生体内の癌組織を反映していることが証明されたことになり、今後腫瘍微小環境内での詳細な分子メカニズムの解明のための協力なツールになると期待できる。
• Foundational approach for next-generation personalized cancer therapywith molecular target agents

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

  Date (from‐to) : 2011 -2012 

  Author : OHBA Yusuke

   

  With the use of a biosensor based on the principle of Forster resonance energy transfer, we isolated imatinib-resistant cells of Ph-positive acute lymphoid leukemia and explored their properties. Twenty-four signaling pathways were found activated in the resistant cells, among which inhibition of the mevalonate pathway was revealed effective in preventing repopulation of such resistant cells. These results thereby indicate that the combined application of statin may serve to cure Ph-positive acute lymphoid leukemia as well as imatinib-resistant chronic myeloid leukemia.
• Exploitation of gene-targeting therapy based on the cellular and biological features of oral-carcinoma stem cell and tumor microenvironment.

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (A)

  Date (from‐to) : 2008 -2011 

  Author : TOTSUKA Yasunori, SHINDOH Masanobu, HIGASHINO Fumihiro, HIDA Kyoko, KITAMURA Tetsuya, OHBA Yusuke, MATSUMOTO Kenichi, OHIRO Yoichi

   

  We have shown that the tumor endothelial cells which exist in cancer stroma have a different gene background from normal endothelial cells. It has been becoming clear that epithelial-mesenchymal transition (EMT) plays an important role in tumor invasion and metastasis. It is expected that the investigation on the interaction of the oral squamous cell carcinoma cell in epithelium nature and a mesenchymal cell will contribute to the establishment of effective therapeutic method because stromal cells have the ability to supply nutrition and oxygen to a tumor. HuR protein usually exists in the nucleus, and HuR is participating to stabilize AU-rich element (ARE) mRNA. We showed that ARE mRNA is stabilized through HuR by a viral carcinogenesis system that concerns cell transformation. Oral environment has a unique features including distinct organs such as salivary glands. We identified that RANKL, an osteoclast inducer, expression was higher in oral environment. Furthermore, when oral cancer cells were implanted in oral region of nude mice, cancer cells proliferated intentionally, and EMT was observed. Tumor microenvironment is deeply correlated with cancer cell proliferation and invasion/metastasis. We have isolated endothelial cells in tumor stroma, and investigated the biological feature of tumor endothelial cells. As a result, tumor angiogenesis was suppressed by inhibiting Cox-2 known also for the factor inducing inflammation, and the resistance for chemothrapy was acquired by MDR1 upregulatin caused by VEGF signaling
• 癌細胞の微小環境適応因子の探索

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

  Date (from‐to) : 2009 -2010 

  Author : 大場 雄介

   

  前年度に引き続き口腔癌をモデルに癌細胞微小環境適応因子として同定したRANKLの機能解析を行りた。RANKLのmRNAレベルおよびタンパクレベルでの発現をRT-PCRと免疫染色で行ったところ、いずれのヒト口腔癌組織における発現が培養細胞に比べて高く、なかでも低分化型の組織での発現が高かった。各種口腔癌細胞株における低いRANKLの発現量は、樹立した元の腫瘍の組織型が低分化型であっても同様であったが、培養癌細胞をヌードマウスの口腔に移植したところ、RANKLの発現が亢進した。一方で同じ細胞株をマウスの下肢に移植したところ、口腔に移植して形成された腫瘍と比較して有意に小さく、RANKLの発現も低かった。すなわち生体内の周囲組織依存的に発現したRANKLが、腫瘍形成を制御することが明らかになった。さらにRANKLの腫瘍形成における役割を明らかにするために、RANKL過剰発現細胞を樹立しマウスの下肢に接種したところ、口腔環境同様に腫瘍の形成が認められた。また組織学的に、コントロール細胞で形成された腫瘍が高分化型扁平上皮癌の組織型を示したのに対し、RANKL発現細胞によって形成された腫瘍は低分化性であった。以上の結果から、RANKLは生体内特異的に発現し腫瘍形成を制御する環境適応因子であり、低分化型の癌すなわち悪性度の高い癌の形成に関与する因子であることが明らかとなった。またこの組織型の変化は上皮間葉転換EMTによることも解明した。 また、RANKLの下流で誘導される因子としてインテグリンα2を同定し、現在悪性度との関与を解析中である。興味深いことにインテグリンα2の発現上昇は活性化型インテグリンの細胞内輸送を亢進するというデータが得られている。近年、EMT等の癌細胞の形態変化と細胞内膜輸送の関連性が報告されつつあり、我々のデータは新たな分子メカニズムを提供する可能性が示唆される。
• Visualization of molecular dynamics of epithelial-mesenchymal and mesenchymal-epithelial transition

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)

  Date (from‐to) : 2008 -2010 

  Author : OHBA Yusuke, TSUDA Masumi

   

  We have hereby identified transcription factor 8 (TCF8) as a regulator of epithelial-mesenchymal transition, a critical process in cancer invasion/metastasis and endothelial mobilization to tumor angiogenesis. The molecular mechanisms by which TCF8 governs EMT were also revealed in both cancer cells and endothelial cells. In addition, novel evidence that the difference in the tumor microenvironment yields distinct feature of cancer malignancy is also provided, which might lead to the development of new strategies to cancer development.
• 蛍光イメージング技術の臨床応用

  共同研究

  Date (from‐to) : 2010
• Clinical application of fluorescence imaging

  Cooperative Research

  Date (from‐to) : 2010
• Informatory expression system connecting cancer and immunity.

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research on Priority Areas

  Date (from‐to) : 2005 -2009 

  Author : TANIGUCHI Tadatsugu, TAKAOKA Akinori, TAMURA Tonohiko, HONDA Kenya, OOBA Yuusuke, YANAI Hideyuki

   

  We investigated biological mechanisms and role of effecter molecules, e.g. interferons, IRFs and Noxa. It revealed that these molecules are involved in transformation, apoptosis induction and metastasis signaling pathways. We also found the cross-talk pathway between TLRs signaling. Moreover, we identified DAI and HMGB1, 2, 3 are important for the nucleic-acid mediated innate immune responses. Since these pathways regulate tumorgenesis and exacerbation, our findings will be proved to be useful for the development of novel tumor therapy in near future.
• Regulatory signaling in intestinal dendritic cells preventing inflammatory bowel diseases

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)

  Date (from‐to) : 2007 -2008 

  Author : HONDA Kenya, TAKEDA Kiyoshi, TAYA Chouji, OHBA Yusuke

   

  腸管樹状細胞を機能的に細分化し、シグナル伝達機構を明らかにし、炎症性腸疾患との関わりを検討することを目的として研究を推進した。新たにCD70が活性化型と抑制型樹状細胞を区別するバイオマーカーとして利用できることが明らかにした。CD70は消化管粘膜固有層特異的に存在する樹状細胞集団の一部において高発現しており、他の臓器にはそのような細胞を認めなかった。更にCD70陽性粘膜固有層樹状細胞は、細胞外ATPの受容体であるP2XやP2Y受容体を高発現しており、ATP刺激によって、共培養したナイーブCD4T細胞をIL17を高産生するTH17細胞へと分化させることも見出した。即ちCD70陽性粘膜固有層樹状細胞は"活性化型樹状細胞"であると考えられた。
• 口腔がんの顎骨浸潤は抑制できるか?-EGFレセプターリン酸化阻害による効果-

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

  Date (from‐to) : 2007 -2008 

  Author : 戸塚 靖則, 進藤 正信, 大場 雄介, 北村 哲也, 樋田 京子, 東野 史裕, 津田 真寿美

   

  口腔がんの多くは舌や歯肉などの口腔内組織に発症するが、これらの臓器・組織は解剖学的に顎骨に近接しており、腫瘍の増大により容易に顎骨に浸潤する。この場合は、腫瘍の根治性の点から、顎骨離断術や部分切断術を施行せざるをえず、顔面の審美性や機能性に多大な障害を生じ、治癒後の社会生活やQOLに大きな影響を及ぼす。それ故、腫瘍の顎骨浸潤を抑制することが可能となれば、口腔がん患者にどって大きな福音となり、その開発が真に望まれている。 口腔癌細胞の多くは、epidemal growth factor (EGF) receptorを高頻度に発現しており、また顎下腺並びに唾液中には高濃度のEGFが存在していることが明らかになっている。従って、口腔癌の微小環境中に存在するEGFが、癌細胞に対してパラクライン的に作用し、骨浸潤に関与する形質発現に重要な影響を及ぼしていることが示唆される。 口腔がん細胞株HSC2, HSC3, HSC4, Ca9.22, SASを用いてEGFRの発現をWestern Blotで検討したところ、全てのがん細胞株でEGFRの発現亢進がみられた。これらの細胞をEGFで刺激した際、EGFRのリン酸化の亢進およびMAPKであるERK1/2, p38.JNKの活性化がみられた。ERK1/2はEGF依存的にPTHrPを発現亢進した。HSC2細胞にPTHrPのsiRNAを導入することで細胞増殖、浸潤活性の抑制がみられた。 このような所見は、口腔がん細胞の顎骨浸潤にはEGF/EGFRによるPTHrPの発現が重要な役割を演じていることを示唆するものだった。
• 受容体サブタイプのモーダルシフトによるアンギオテンシン感受性調節機構の解明

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research on Priority Areas

  Date (from‐to) : 2007 -2008 

  Author : 大場 雄介

   

  レニン-アンギオテンシン系の研究は国内外を問わずもっともホットな領域の一つといえる。アンジオテンシンは二つのセルセンサーAT1とAT2を介してシグナルを伝えることが知られているが、AT1については既に多くの知見が得られているのに対し、AT2の機能は未知のままである。我々はAT2の発現がAT1シグナルに制御されているという基礎的データを得ており、細胞はAT1からAT2へセンサーをモーダルシフトすることにより、外界に存在するアンジオテンシンのセンシング機構を制御しているものと考えられる、その分子機序が解明されればインパクトは大きい。 本研究ではAT1がアンジオテンシンをセンスして惹起されるERKの活性化を指標にAT1とAT2の機能連関の解明を試みた。AT2はAT2に対し、ERKのリン酸化に関しては拮抗的に働くが、この作用は他のEGFやLPAによるERKのリン酸化に対しては認められないことから、シグナル伝達の極めて上流でクロストークしていることが示唆された。また、蛍光タグを付加したAT1とAT2を発現させ、蛍光顕微鏡で観察したところ、両者が共発現した場合には互いの局在を変化させ共局在することが明らかになった。そこで、2つのセルセンサーの結合を解析したところ、免疫沈降法および蛍光共鳴エネルギー移動(FRET) 法の双方でAT1とAT2のアンジオテンシン依存的な結合が認められた。特にFRETを用いたイメージングでは2つのセルセンサーが細胞膜上で結合した後に、エンドサイトーシスされ、エンドソーム上で結合することが、両者センサーからのシグナルクロストークに重要であることが明らかとなった。これらの現象には、双方の受容体にリガンドが結合することが必要であり、更にその下流に位置する因子として、あるセリンスレオニンキナーゼの必要性を同定した。
• 3分子複合体を生きた細胞で検出する技術の確立

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

  Date (from‐to) : 2007 -2008 

  Author : 大場 雄介

   

  生きた細胞において多分子複合体が、何時何処で形成されるかを追及することは、生命をシステムと理解し、正常状態や病態発症メカニズムを解明する上で重要であにも関わらず、この重要なメカニズムを検出する実験系そのものが現在のところ確立されていない。本研究課題では、生きた細胞における多分子複合体の検出系を確立し、新時代の細胞内情報伝達研究推進への足がかりを築くことを目的として研究を開始した。まず、淡色で分子間相互作用の観察を可能とする、蛍光蛋白質再構成(BiFC)法を用いて蛋白質間相互作用の動的観察系を世界に先がかけて開発した。この技術を用い、RasとPI3Kによる特異的なエンドゾーム上での結合を画像化し、その動きを追跡することに成功した。更にこの研究の過程で、実際にBiFCと他の蛍光蛋白質および、免疫染色を併用することにより、Ras、PI3Kの結合とその代謝産物PIP_3の産生、エンドゾームマーカーであるEEA1の局在からなる、合計3つの蛋白質と一つの脂質の共局在を可視化することに成功した。それらの分子メカニズムを詳細に解析する過程で、RasによるPI3Kの活性化には二つの細胞内シグナル伝達経路が存在し、ひとつはRas非依存性、もう一つはRas依存性であり、後者がエンドゾームにおけるPI3Kの活性化に必要であることも示した。PI3Kの活性化は細胞の生存に必須であることから、その機能阻害は細胞障害が懸念されるが、今回得られた結果は、PI3Kの活性を時空間的に変調することでその一部の機能を調節1可能であることを示唆しており、エンドサイトーシスを介した種々の物質の取り込みを制御する新たな手法の開発に発展すると期待される。
• 腫瘍細胞ー微小環境相互作用の時空間ダイナミクス

  科学研究費補助金

  Date (from‐to) : 2008
• Spatiotemporal dynamics of tumor cell-microenvironment interaction

  Grant-in-Aid for Scientific Research

  Date (from‐to) : 2008
• 多次元バイオイメージングで解明するがんと免疫の時空間シグナルネットワーク

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Young Scientists (A)

  Date (from‐to) : 2005 -2007 

  Author : 大場 雄介

   

  1.癌における時空間シグナルネットワーク (1)口腔内は上皮細胞増殖因子(EGF)が多量に存在する環境であるため、口腔癌の悪性化にはEGF受容体下流の情報伝達経路が重要である。口腔癌細胞において、EGF受容体の下流で上皮小体ホルモン関連蛋白質(PTHrP)が誘導され、PTHrPが口腔癌細胞の細胞増殖、運動能及び浸潤能等の悪性度を制御していることを明らかにした。本研究成果はBiochem. Biophys. Res. Commun.誌に発表した。 (2)悪性膠芽腫細胞における浸潤能を制御する分子機構として、転写因子OLIG2の発現と低分子量G蛋白質Rhoの活性化、さらにRhoの活性化の局在との関連を明らかにした。本研究成果はMol. Cancer Res.誌に発表した。 2.免疫におけるシグナルネットワーク (1)核酸による自然免疫系発動に関する分子動態について明らかにした。DNAをセンスする細胞内因子DAIを同定し、DAIとB型DNAの直接結合を画像化することに成功した。また、その過程が培養細胞でのインターフェロン産生に重要であることを明らかにし、Natureに報告した。また、2重鎖RNAに応答するToll様受容体下流におけるアダプター分子TICAM1の細胞内動態について解析し、J. Immunolo.誌に報告した。 (2)インターフェロン産生における負の制御因子IRF2のノックアウトマウスを用い、過剰なインターフェロンシグナルによって生じる慢性貧血の分子機序を明らかにした。本研究成果は、Exp. Hematol.誌に発表した。 本研究課題3年間での成果について、英文学術雑誌に計13本の報告を行った。
• 蛍光バイオイメージングを用いた細胞内情報伝達の時空間制御機構の解明

  科学研究費補助金

  Date (from‐to) : 2006
• Spatiotemporal regulation of signal transduction pathways using fluorescence imaging

  Grant-in-Aid for Scientific Research

  Date (from‐to) : 2006
• Rasファミリー蛋白質Ralの活生化を生きた細胞内で観察し、その機能を解明する

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Young Scientists (B)

  Date (from‐to) : 2002 -2003 

  Author : 大場 雄介

   

  RalはRasファミリーGたんぱく質の一つで、Rasにより活性化される。最近、エンドサイトーシス、エキソサイトーシス、フィロポディアの形成に対するRalの関与が示唆されつつあるものの、詳細な分子機序は未知の点が多い。そこで生きた細胞内でRal活性化の時空間的な制御機構の解析を通じて、Ralの生理的機能を知るために、蛍光共鳴エネルギー移動(FRET)の原理を利用したモニター分子を作製し解析した。 モニター分子の構造はYFP、RalA、RalBP、CFP及び膜局在化配列からなる。この分子を発現する293T細胞可溶化液のFRET効率を蛍光分光光度計で測定したところ、GTP結合率とFRET効率が正の相関を示した。従って作製したモニター分子はRalの活性化をFRET効率の上昇として検出できることが確認できた。次にCOS1細胞において上皮細胞増殖因子(EGF)依存性のRalの活性化を多重蛍光タイムラプス顕微鏡で観察し、FRET効率を画像化して解析したところ、EGFによって生じる葉状仮足に限局してRalが活性化していた。この結果はRalの活生化に関し初めて得られた時空間情報である。 さらに、EGF刺激によるRal活性化の機構を詳細に検討した。Rasの優勢劣性型変異体を発現した細胞では、Ralの活性化が抑制されていることから、EGF依存性のRal活性化にはRasの活性化を必要とすることが確認された。しかし、Rasの活性化は葉状仮足で高いが、必ずしも葉状仮足に限局するわけではないので、葉状仮足の形成もまたRalの活性化に必要であることが示唆された。実際、優勢劣性型変異体のRacを導入して、EGF依存性葉状仮足形成を阻害すると、Ralの活性化は阻害された。すなわち、増殖因子依存性Ralの活性化は複数の経路によって制御されていることが明らかとなった。
• 低分子量G蛋白が細胞膜で情報を受け取るメカニズムの可視化

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research on Priority Areas

  Date (from‐to) : 1999 -2003 

  Author : 松田 道行, 黒川 量雄, 大場 雄介, 中村 岳史, 望月 直樹

   

  本研究の目的は、細胞内情報伝達の重要なキープレーヤーである低分子量G蛋白の活性化の様子を生細胞でリアルタイムにモニターするプローブを作成し、バーチャル細胞作成のために必要な、細胞内情報伝達の時空間パラメータを獲得することである。本年度は、K-Ras、N-Ras、R-Ras、RalA、RalBのプローブ開発を行った。K-Ras、N-Ras、RalAに関しては感度の高いプローブの開発に成功したが、R-RasおよびRalBのプローブは感度が低く、改良の余地を残している。また、既存のプローブの高感度化をYFPおよびCFPをより明るいものに置換することにより達成した。これにより、画像データのシグナルノイズ比を向上させることに成功した。さらに、共焦点レーザー顕微鏡を用いて、細胞内の膜画分と細胞膜画分とのシグナルを定量的に分ける手法を確立した。 一方、これら新規に開発したプローブを使って様々な生命現象の解明に取り組んだ。特に、これまで機能の不明であったRalAが、細胞増殖因子の刺激により葉状突起で限局して活性化されること、RalAの活性化が増殖因子依存性あるいは細胞運動時の葉状突起の形成に必要であることをさまざまな手法を用いて証明した。RalAは小胞の融合に関与するとうい他グループのデータとあわせて考えると、葉状突起の形成には、細胞骨格系と細胞膜系の双方のダイナミックな変化が必要であり、RhoファミリーG蛋白はその前者に、RalAはその後者に密接に関わることが示唆された。

Industrial Property Rights

• 特開2009-278942:BCR−ABLチロシンキナーゼ活性測定用試薬  2009/12/03

  大場 雄介, 近藤 健  国立大学法人 北海道大学  200903026427616457
• 特開2008-148556:インターフェロン−α及び/又はβ(IFN−α/β)の発現誘導を促進する補助剤のスクリーニングする方法  2008/07/03

  谷口 維紹, 本田 賢也, 大場 雄介  国立大学法人 東京大学  200903046155691471
• AGENT FOR INHIBITING VIRAL INFECTION AND/OR TREATING INFECTIOUS DISEASE, AND METHOD FOR INHIBITING VIRAL INFECTION AND/OR TREATING INFECTIOUS DISEASE

  WO/2011/126071
• METHOD FOR SCREENING OF SUBSTANCE CAPABLE OF PREVENTING VIRUS INFECTION, AND AGENT FOR PREVENTING VIRUS INFECTION

  WO/2011/046160
• ANTI-ANGIOGENIC AGENT

  WO 2010/005046
• ＢＣＲ－ＡＢＬチロシンキナーゼ活性測定試薬

  2009-278942