Details of a Researcher - Saya, Hideyuki

Papers - Saya, Hideyuki

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Regulation of MKL1 via actin cytoskeleton dynamics drives adipocyte differentiation

Nobusue Hiroyuki, Onishi Nobuyuki, Shimizu Takatsune, Sugihara Eiji, Oki Yoshinao, Sumikawa Yuko, Chiyoda Tatsuyuki, Akashi Koichi, Saya Hideyuki, Kano Koichiro

Nature Communications 5 3368 2014

Cellular differentiation is regulated through activation and repression of defined transcription factors. A hallmark of differentiation is a pronounced change in cell shape, which is determined by dynamics of the actin cytoskeleton. Here we show that regulation of the transcriptional coactivator MKL1 (megakaryoblastic leukemia 1) by actin cytoskeleton dynamics drives adipocyte differentiation mediated by peroxisome proliferator-activated receptor γ (PPARγ), a master transcriptional regulator of adipogenesis. Induction of adipocyte differentiation results in disruption of actin stress fibres through downregulation of RhoA-ROCK signalling. The consequent rapid increase in monomeric G-actin leads to the interaction of G-actin with MKL1, which prevents nuclear translocation of MKL1 and allows expression of PPARγ followed by adipogenic differentiation. Moreover, we found that MKL1 and PPARγ act in a mutually antagonistic manner in the adipocytic differentiation programme. Our findings thus provide new mechanistic insight into the relation between the dynamics of cell shape and transcriptional regulation during cellular differentiation.

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Basic biology of gliomas

Saya Hideyuki, Shibao Shunsuke, Sampetrean Oltea

Japanese Journal of Neurosurgery 23 ( 7 ) 541 - 546 2014

ISSN 0917-950X

Gliomas are a group of primary brain neoplasms classified mainly on the basis of their morphological resemblance to glial cells. Their most malignant form, grade IV glioma or glioblastoma multiforme, continues to remain lethal, partly due to a pronounced intratumoral heterogeneity which prevents effective therapeutic targeting. The heterogeneity seen in glioblastomas ranges from multiple genetic and epigenetic alterations to a cellular diversity that has recently been traced to the existence of tumor stem cells.Glioma stem cells, like other types of cancer stem cells, have the ability to self-renew and differentiate into multiple tissue-specific lineages. They can therefore maintain and propagate the tumor by continuously giving rise to a variety of malignant offspring and also initiate tumor recurrence from a very small number of cells. Furthermore, they are also considered to be the cells most likely to survive even multi-modality treatments, due to characteristics such as slow cycling or quiescence, high drug efflux capacity and strong antioxidant and immune defense systems.With the addition of each new research finding, the concept of glioma stem cell itself continues to evolve. However, many aspects of glioma stem cell biology are still insufficiently understood. It is not known which cells they originate from, what their relationship to normal tissue stem cells is and what mechanisms or events underlie their malignant transformation. Furthermore, whether the answers to these questions are the same for all glioma stem cells is also a matter that requires further investigation.This review focuses on our current understanding of glioma stem cells, the signaling pathways behind their pathogenic features and their role in glioma biology. We also discuss possible answers to the questions mentioned above, in the context of knowledge obtained from the study of tumor stem cells of several other types of malignancies. Finally, we review the importance of glioma stem cells in treatment resistance in glioblastoma and the implications of stem cell-specific targeting strategies.

A human cancer xenograft model utilizing normal pancreatic duct epithelial cells conditionally transformed with defined oncogenes

Inagawa Yuki, Yamada Kenji, Yugawa Takashi, Ohno Shin Ichi, Hiraoka Nobuyoshi, Esaki Minoru, Shibata Tatsuhiro, Aoki Kazunori, Saya Hideyuki, Kiyono Tohru

Carcinogenesis 35 ( 8 ) 2014

ISSN 0143-3334

Pancreatic ductal adenocarcinomas (PDACs) are considered to arise through neoplastic transformation of human pancreatic duct epithelial cells (HPDECs). In order to evaluate the biological significance of genetic and epigenetic alterations in PDACs, we isolated primary HPDECs and established an in vitro carcinogenesis model. Firstly, lentivirus-mediated transduction of KRAS^G12V, MYC and human papillomavirus 16 (HPV16) E6/E7 under the control of a tetracyclin-inducible promoter efficiently immortalized and transformed primary HPDECs, which gave rise to adenocarcinomas subcutaneously in an immune-deficient mouse xenograft model, depending on expression of the four genes. The tumors regressed promptly upon shutting-off the oncogenes, and the remaining tissues showed histological features corresponding to normal ductal structures with simple columnar epithelium. Re-expression of the oncogenes resulted in development of multiple PDACs through pancreatic intraepithelial neoplasia-like structures. We also succeeded in efficient immortalization of primary HPDECs with transduction of mutant CDK4, cyclin D1 and TERT. The cells maintained a normal diploid status and formed duct-like structures in a three dimensional culture. In combination with p53 silencing, KRAS^G12V alone was sufficient to fully transform the immortalized HPDECs, and MYC markedly accelerated the development of tumors. Our PDAC model supports critical roles of KRAS mutations, inactivation of the p53 and p16-pRB pathways, active telomerase and MYC expression in pancreatic carcinogenesis and thus recapitulates many features of human PDAC development. The present system with reversible control of oncogene expression enabled de novo development of PDAC from quasi-normal human tissues pre-formed subcutaneously in mice and might be applicable to carcinogenesis models in many organ sites.

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Tumor necrosis factor-α modulates epithelial mesenchymal transition mediators ZEB2 and S100A4 to promote cholangiocarcinoma progression

Techasen Anchalee, Namwat Nisana, Loilome Watcharin, Duangkumpha Kassaporn, Puapairoj Anucha, Saya Hideyuki, Yongvanit Puangrat

Journal of Hepato-Biliary-Pancreatic Sciences 21 ( 9 ) 703 - 711 2014

ISSN 1868-6974

Background The epithelial-mesenchymal transition (EMT) process strongly contributes to cancer metastasis. This study was to investigate the alteration of EMT-related proteins (ZEB1, ZEB2 and S100A4) in cholangiocarcinoma (CCA) tissues. The effect of tumor necrosis factor-α (TNF-α) on the expression of those molecules in CCA cells was investigated. Methods The quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assay was used to quantify ZEB1, ZEB2 and S100A4 mRNA levels in 50 CCA tissues and related its expression to clinicopathological data. ZEB2 protein immunostaining was investigated in 165 CCA tissues. The effect of TNF-α on EMT-related CCA cell migration was evaluated using qRT-PCR, immunofluorescence and transwell migration assays. Results ZEB2 and S100A4 mRNA levels were found to be higher in CCA tissues. High levels of S100A4 mRNA and ZEB2 protein were significantly associated with CCA metastasis (P = 0.04 and P = 0.03). Moreover, a trend toward statistical association was found with high levels of both ZEB2 mRNA and protein with shorter survival time (P = 0.10 and P = 0.19). In addition, TNF-α induced CCA cell migration by the induction of transforming growth factor-β (TGF-β) resulting in ZEB2 and S100A4 mRNA and protein activation. Conclusions These studies demonstrate that TNF-α plays crucial role in the progression of CCA by activating TGF-β signaling and the induction of ZEB2 and S100A4, EMT-related proteins expression.

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Integrated analysis identifies different metabolic signatures for tumor-initiating cells in a murine glioblastoma model

Saga Isako, Shibao Shunsuke, Okubo Jun, Osuka Satoru, Kobayashi Yusuke, Yamada Sachiko, Fujita Satoshi, Urakami Kenichi, Kusuhara Masatoshi, Yoshida Kazunari, Saya Hideyuki, Sampetrean Oltea

Neuro-Oncology 16 ( 8 ) 1048 - 1056 2014

ISSN 1522-8517

Background The metabolic preference of malignant glioma for glycolysis as an energy source is a potential therapeutic target. As a result of the cellular heterogeneity of these tumors, however, the relation between glycolytic preference, tumor formation, and tumor cell clonogenicity has remained unknown. To address this issue, we analyzed the metabolic profiles of isogenic glioma-initiating cells (GICs) in a mouse model. Methods GICs were established by overexpression of H-RasV12 in Ink4a/Arf-null neural stem cells. Subpopulations of these cells were obtained by single-cell cloning, and clones differing in extracellular acidification potential were assessed for metabolic characteristics. Tumors formed after intracranial implantation of these clones in mice were examined for pathological features of glioma and expression of glycolytic enzymes. Results Malignant transformation of neural stem cells resulted in a shift in metabolism characterized by an increase in lactic acid production. However, isogenic clonal populations of GICs manifested pronounced differences in glucose and oxygen consumption, lactate production, and nucleoside levels. These differences were paralleled by differential expression of glycolytic enzymes such as hexokinase 2 and pyruvate kinase M2, with this differential expression also being evident in tumors formed by these clones in vivo. Conclusions The metabolic characteristics of glioma cells appear early during malignant transformation and persist until the late stages of tumor formation. Even isogenic clones may be heterogeneous in terms of metabolic features, however, suggesting that a more detailed understanding of the metabolic profile of glioma is imperative for effective therapeutic targeting.

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Katanin p60 contributes to microtubule instability around the midbody and facilitates cytokinesis in rat cells

Matsuo Moe, Shimodaira Tetsuhiro, Kasama Takashi, Hata Yukie, Echigo Ayumi, Okabe Masaki, Arai Kazuya, Makino Yasutaka, Niwa Shin Ichiro, Saya Hideyuki, Kishimoto Toshihiko

PLoS One 8 ( 11 ) 2013.11

The completion of cytokinesis is crucial for mitotic cell division. Cleavage furrow ingression is followed by the breaking and resealing of the intercellular bridge, but the detailed mechanism underlying this phenomenon remains unknown. Katanin is a microtubule-severing protein comprised of an AAA ATPase subunit and an accessory subunit designated as p60 and p80, respectively. Localization of katanin p60 was observed at the midzone to midbody from anaphase to cytokinesis in rat cells, and showed a ring-shaped distribution in the gap between the inside of the contractile ring and the central spindle bundle in telophase. Katanin p60 did not bind with p80 at the midzone or midbody, and localization was shown to be dependent on microtubules. At the central spindle and the midbody, no microtubule growth plus termini were seen with katanin p60, and microtubule density was inversely correlated with katanin p60 density in the region of katanin p60 localization that seemed to lead to microtubule destabilization at the midbody. Inhibition of katanin p60 resulted in incomplete cytokinesis by regression and thus caused the appearance of binucleate cells. These results suggest that katanin p60 contributes to microtubule instability at the midzone and midbody and facilitates cytokinesis in rat cells.

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Redox regulation in stem-like cancer cells by CD44 variant isoforms

Nagano O., Okazaki S., Saya H.

Oncogene 32 ( 44 ) 5191 - 5198 2013.10

ISSN 0950-9232

Increasing evidence indicates that several types of solid tumor are hierarchically organized and sustained by a distinct population of cancer stem cells (CSCs). CSCs possess enhanced mechanisms of protection from stress induced by reactive oxygen species (ROS) that render them resistant to chemo-and radiotherapy. Expression of CD44, especially variant isoforms (CD44v) of this major CSC marker, contributes to ROS defense through upregulation of the synthesis of reduced glutathione (GSH), the primary intracellular antioxidant. CD44v interacts with and stabilizes xCT, a subunit of the cystine-glutamate transporter xc(-), and thereby promotes cystine uptake for GSH synthesis. Given that cancer cells are often exposed to high levels of ROS during tumor progression, the ability to avoid the consequences of such exposure is required for cancer cell survival and propagation in vivo. CSCs, in which defense against ROS is enhanced by CD44v are thus thought to drive tumor growth, chemoresistance and metastasis. Therapy targeted to the CD44v-xCT system may therefore impair the ROS defense ability of CSCs and thereby sensitize them to currently available treatments.

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Characteristics of glioma stem cells

Sampetrean Oltea, Saya Hideyuki

Brain Tumor Pathology 30 ( 4 ) 209 - 214 2013.10

ISSN 1433-7398

The cancer stem cell theory postulates that tumors are sustained by a select cell population with specific features, such as self-renewal ability and the capacity to give rise to a heterogeneous mass of tumor cells. The existence of such cells has been demonstrated for glioblastoma, with these cells being referred to as glioma stem cells (GSCs). Glioblastomas are notoriously heterogeneous tumors, however, and the isolation and characterization of their stem cells will require further investigations. Furthermore, the lack of unequivocal markers for GSCs and a partial overlap in characteristics with other cells often lead to confusion. Here, we review the characteristics necessary for a glioma cell to be considered a stem cell, and we adopt our murine glioblastoma model based on genetically modified neural stem cells to illustrate and discuss the GSC concept.

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Functional role of CD44v-xCT system in the development of spasmolytic polypeptide-expressing metaplasia

Wada Takeyuki, Ishimoto Takatsugu, Seishima Ryo, Tsuchihashi Kenji, Yoshikawa Momoko, Oshima Hiroko, Oshima Masanobu, Masuko Takashi, Wright Nicholas A., Furuhashi Satoshi, Hirashima Kotaro, Baba Hideo, Kitagawa Yuko, Saya Hideyuki, Nagano Osamu

Cancer Science 104 ( 10 ) 1323 - 1329 2013.10

ISSN 1347-9032

Cancer development is often preceded by the appearance of preneoplastic lesions. In gastric carcinogenesis, chronic inflammation and histopathologic progression of the stomach epithelium lead to the development of metaplasia and eventually adenocarcinoma. The cell surface protein CD44, especially its variant isoforms (CD44v), has been implicated in metaplasia-carcinoma sequence progression in the stomach. We recently found that CD44v interacts with and stabilizes xCT, a subunit of the cystine transporter system xc(-), in cancer cells and thereby increases cystine uptake and confers resistance to various types of cellular stress in vivo. The functional relevance of CD44v and xCT in the development of preneoplastic lesions, however, has remained unknown. We have now examined the role of the CD44v-xCT system in the development of spasmolytic polypeptide-expressing metaplasia (SPEM) in mouse models of gastric carcinogenesis. CD44v was found to be expressed de novo in SPEM, and CD44v⁺ metaplastic cells manifested upregulation of xCT expression compared with CD44v^- cells. Genetic ablation of CD44 or treatment with sulfasalazine, an inhibitor of xCT-dependent cystine transport, suppressed the development of SPEM and subsequent gastric tumor growth. Therapy targeted to CD44v-xCT could thus prove effective for prevention or attenuation of the CD44v-dependent development of preneoplastic lesions and cancer.

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Statins Decrease Lung Inflammation in Mice by Upregulating Tetraspanin CD9 in Macrophages

Jin Yingji, Tachibana Isao, Takeda Yoshito, He Ping, Kang Sujin, Suzuki Mayumi, Kuhara Hanako, Tetsumoto Satoshi, Tsujino Kazuyuki, Minami Toshiyuki, Iwasaki Takeo, Nakanishi Kaori, Kohmo Satoshi, Hirata Haruhiko, Takahashi Ryo, Inoue Koji, Nagatomo Izumi, Kida Hiroshi, Kijima Takashi, Ito Mari, Saya Hideyuki, Kumanogoh Atsushi

PLoS One 8 ( 9 ) 2013.09

Tetraspanins organize protein complexes in tetraspanin-enriched membrane microdomains that are distinct from lipid rafts. Our previous studies suggested that reduction in the levels of tetraspanins CD9 and CD81 may be involved in the progression of inflammatory lung diseases, especially COPD. To search for agents that increase the levels of these tetraspanins, we screened 1,165 drugs in clinical use and found that statins upregulate CD9 and CD81 in RAW264.7 macrophages. The lipophilic statins, fluvastatin and simvastatin, reversed LPS-induced downregulation of CD9 and CD81, simultaneously preventing TNF-α and matrix metalloproteinase-9 production and spreading of RAW264.7 cells. These statins exerted anti-inflammatory effects in vitro in wild-type macrophages but not in CD9 knockout macrophages, and decreased lung inflammation in vivo in wild-type mice but not in CD9 knockout mice, suggesting that their effects are dependent on CD9. Mechanistically, the statins promoted reverse transfer of the LPS-signaling mediator CD14 from lipid rafts into CD9-enriched microdomains, thereby preventing LPS receptor formation. Finally, upregulation of CD9/CD81 by statins was related to blockade of GTPase geranylgeranylation in the mevalonate pathway. Our data underscore the importance of the negative regulator CD9 in lung inflammation, and suggest that statins exert anti-inflammatory effects by upregulating tetraspanin CD9 in macrophages.

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Three-dimensional culture of sebaceous gland cells revealing the role of prostaglandin E₂-induced activation of canonical Wnt signaling

Yoshida Go J., Saya Hideyuki

Biochemical and Biophysical Research Communications 438 ( 4 ) 640 - 646 2013.09

ISSN 0006-291X

Background: Prostaglandin E₂ (PGE₂) is a proinflammatory mediator and activates the canonical Wnt-β-catenin signaling pathway in hematopoietic stem cells. The SZ95 cell line was established from human sebaceous gland cells and is studied as a model system for these cells. Given that 2D culture of SZ95 cells does not recapitulate the organization of sebaceous glands in situ, we developed a 3D culture system for these cells and examined the effects of PGE₂ on cell morphology and function. Results: SZ95 cells maintained in 3D culture formed organoids that mimicked the organization of sebaceous glands in situ, including the establishment of a basement membrane. Organoids exposed to PGE₂ were larger and adopted a more complex organization compared with control organoids. PGE₂ activated the canonical Wnt signaling pathway as well as increased cell viability and proliferation, mitochondrial metabolism, and lipid synthesis in the organoids. Conclusions: Culture of SZ95 cells in 3D culture system recapitulates the structure and susceptibility to PGE₂ of sebaceous glands in situ and should prove useful for studies of the response of these glands to inflammation and other environmental stressors. Our results also implicate PGE₂-induced activation of canonical Wnt signaling pathway in regulation of the morphology,proliferation, and function of "semi-vivo" sebaceous glands.

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Energy management by enhanced glycolysis in G₁-phase in human colon cancer cells in vitro and in vivo

Bao Yan, Mukai Kuniaki, Hishiki Takako, Kubo Akiko, Ohmura Mitsuyo, Sugiura Yuki, Matsuura Tomomi, Nagahata Yoshiko, Hayakawa Noriyo, Yamamoto Takehiro, Fukuda Ryo, Saya Hideyuki, Suematsu Makoto, Minamishima Yoji Andrew

Molecular Cancer Research 11 ( 9 ) 973 - 985 2013.09

ISSN 1541-7786

Activation of aerobic glycolysis in cancer cells is well known as theWarburg effect, although its relation to cellcycle progression remains unknown. In this study, human colon cancer cells were labeled with a cell-cycle phasedependent fluorescent marker Fucci to distinguish cells in G1-phase and those in S⁺G2/M phases. Fucci-labeled cells served as splenic xenograft transplants in super-immunodeficient NOG mice and exhibited multiple metastases in the livers, frozen sections of which were analyzed by semiquantitative microscopic imaging mass spectrometry. Results showed that cells in G1-phase exhibited higher concentrations of ATP, NADH, and UDPN- acetylglucosamine than those in S andG2-Mphases, suggesting accelerated glycolysis inG1-phase cells in vivo. Quantitative determination of metabolites in cells synchronized in S, G2-M, and G1 phases suggested that efflux of lactate was elevated significantly in G1-phase. By contrast, ATP production in G2-Mwas highly dependent on mitochondrial respiration, whereas cells in S-phase mostly exhibited an intermediary energy metabolism between G1 and G2-Mphases. Isogenic cells carrying a p53-null mutation appeared more active in glycolysis throughout the cell cycle than wild-type cells. Thus, as the cell cycle progressed from G2-M toG1 phases, the dependency of energy production on glycolysis was increased while the mitochondrial energy production was reciprocally decreased. Implications: These results shed light on distinct features of the phase-specific phenotypes of metabolic systems in cancer cells.

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Acquired expression of NFATc1 downregulates e-cadherin and promotes cancer cell invasion

Oikawa Tsukasa, Nakamura Atsuko, Onishi Nobuyuki, Yamada Taketo, Matsuo Koichi, Saya Hideyuki

Cancer Research 73 ( 16 ) 5100 - 5109 2013.08

ISSN 0008-5472

NFATc1 is a transcription factor that regulates T-cell development, osteoclastogenesis, and macrophage function. Given that T cells, osteoclasts, and macrophages in the tumor microenvironment are thought to modulate tumor progression, tumor cells may acquire NFATc1 expression through fusion with these NFATc1- expressing normal cells. We here revealed that a small proportion of tumor cells in human carcinoma specimens expressed NFATc1. To investigate the consequences of NFATc1 acquisition by tumor cells, we established A549 and MCF7 cell lines expressing a constitutively active form of NFATc1 (NFATc1CA) in an inducible manner. The expression of NFATc1CA promoted cancer cell invasion in association with changes in cell morphology. Analysis of gene expression andRNAinterference experiments revealed that NFATc1CA suppressed E-cadherin expression by upregulating the transcriptional repressors Snail and Zeb1 in a manner independent of TGF-b signaling. Induced expression of NFATc1CA also downregulated E-cadherin expression and increased invasive activity in tumor xenografts in vivo. Our results thus suggest that the acquisition of NFATc1 expression contributes to tumor progression.

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CD44 variant 9 expression in primary early gastric cancer as a predictive marker for recurrence

Hirata K., Suzuki H., Imaeda H., Matsuzaki J., Tsugawa H., Nagano O., Asakura K., Saya H., Hibi T.

British Journal of Cancer 109 ( 2 ) 379 - 386 2013.07

ISSN 0007-0920

Background:Multiple early gastric cancers (EGCs) may develop in 6-14% of patients even after achieving curative endoscopic submucosal dissection (ESD); however, a useful biomarker for predicting recurrence is not available. The present study investigated whether the expression of CD44 variant 9 (CD44v9), a functional cancer stem cell marker, in the primary gastric cancer tissue represents an indicator of recurrence.Methods:Eighty-eight patients who underwent ESD for EGC from 2008 to 2010 were enrolled and monitored for recurrence for 3 years. The expression levels of CD44v9 in the tissue of initial EGCs were evaluated by immunohistochemistry, and the recurrence rate was compared between CD44v9-positive and CD44v9-negative groups. The mucin phenotype and expression of microRNA-21 (miR-21) and programmed cell death protein 4 (PDCD4) were also analysed.Results:The recurrence rate of EGC was significantly higher in the CD44v9-positive group than in the CD44v9-negative group (hazard ratio (HR), 21.8; 95% confidence interval (CI), 5.71-83.1). However, mucin phenotypes and the expression of miR-21 and PDCD4 did not predict recurrence after ESD. Meanwhile, grade of gastric atrophy was also identified as a significant marker of multiple recurrence (HR, 4.95; 95% CI, 1.30-18.8).Conclusion:CD44 variant 9 expression represents a potential predictive marker for recurrence in EGC.

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Twist2 functions as a tumor suppressor in murine osteosarcoma cells

Ishikawa Tomoki, Shimizu Takatsune, Ueki Arisa, Yamaguchi Sayaka I., Onishi Nobuyuki, Sugihara Eiji, Kuninaka Shinji, Miyamoto Takeshi, Morioka Hideo, Nakayama Robert, Kobayashi Eisuke, Toyama Yoshiaki, Mabuchi Yo, Matsuzaki Yumi, Yamaguchi Rui, Miyano Satoru, Saya Hideyuki

Cancer Science 104 ( 7 ) 880 - 888 2013.07

ISSN 1347-9032

The epithelial-mesenchymal transition (EMT) contributes to the malignant progression of cancer cells including acquisition of the ability to undergo metastasis. However, whereas EMT-related transcription factors (EMT-TF) are known to play an important role in the malignant progression of epithelial tumors, their role in mesenchymal tumors remains largely unknown. We show that expression of the gene for Twist2 is downregulated in human osteosarcoma and correlates inversely with tumorigenic potential in mouse osteosarcoma. Forced expression of Twist2 in highly tumorigenic murine osteosarcoma cells induced a slight inhibition of cell growth in vitro but markedly suppressed tumor formation in vivo. Conversely, knockdown of Twist2 in osteosarcoma cells with a low tumorigenic potential promoted tumor formation in vivo, suggesting that Twist2 functions as a tumor suppressor in osteosarcoma cells. Furthermore, Twist2 induced expression of fibulin-5, which has been reported as a tumor suppressor. Medium conditioned by mouse osteosarcoma cells overexpressing Twist2 inhibited expression of the MMP9 gene as well as invasion in mouse embryonic fibroblasts, and forced expression of Twist2 in osteosarcoma cells suppressed MMP9 gene expression in tumor tissue. Data from the present study suggest that Twist2 inhibits formation of a microenvironment conducive to tumor growth and thereby attenuates tumorigenesis in osteosarcoma.

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Targeting Aurora B to the Equatorial Cortex by MKlp2 Is Required for Cytokinesis

Kitagawa Mayumi, Fung Suet Yin Sarah, Onishi Nobuyuki, Saya Hideyuki, Lee Sang Hyun

PLoS One 8 ( 6 ) 2013.06

Although Aurora B is important in cleavage furrow ingression and completion during cytokinesis, the mechanism by which kinase activity is targeted to the cleavage furrow and the molecule(s) responsible for this process have remained elusive. Here, we demonstrate that an essential mitotic kinesin MKlp2 requires myosin-II for its localization to the equatorial cortex, and this event is required to recruit Aurora B to the equatorial cortex in mammalian cells. This recruitment event is also required to promote the highly focused accumulation of active RhoA at the equatorial cortex and stable ingression of the cleavage furrow in bipolar cytokinesis. Specifically, in drug-induced monopolar cytokinesis, targeting Aurora B to the cell cortex by MKlp2 is essential for cell polarization and furrow formation. Once the furrow has formed, MKlp2 further recruits Aurora B to the growing furrow. This process together with continuous Aurora B kinase activity at the growing furrow is essential for stable furrow propagation and completion. In contrast, a MKlp2 mutant defective in binding myosin-II does not recruit Aurora B to the cell cortex and does not promote furrow formation during monopolar cytokinesis. This mutant is also defective in maintaining the ingressing furrow during bipolar cytokinesis. Together, these findings reveal that targeting Aurora B to the cell cortex (or the equatorial cortex) by MKlp2 is essential for the maintenance of the ingressing furrow for successful cytokinesis.

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CAMP/PKA signalling reinforces the LATS-YAP pathway to fully suppress YAP in response to actin cytoskeletal changes

Kim Minchul, Kim Miju, Lee Seunghee, Kuninaka Shinji, Saya Hideyuki, Lee Ho, Lee Sookyung, Lim Dae Sik

EMBO Journal 32 ( 11 ) 1543 - 1555 2013.05

ISSN 0261-4189

Actin cytoskeletal damage induces inactivation of the oncoprotein YAP (Yes-associated protein). It is known that the serine/threonine kinase LATS (large tumour suppressor) inactivates YAP by phosphorylating its Ser127 and Ser381 residues. However, the events downstream of actin cytoskeletal changes that are involved in the regulation of the LATS-YAP pathway and the mechanism by which LATS differentially phosphorylates YAP on Ser127 and Ser381 in vivo have remained elusive. Here, we show that cyclic AMP (cAMP)-dependent protein kinase (PKA) phosphorylates LATS and thereby enhances its activity sufficiently to phosphorylate YAP on Ser381. We also found that PKA activity is involved in all contexts previously reported to trigger the LATS-YAP pathway, including actin cytoskeletal damage, G-protein-coupled receptor activation, and engagement of the Hippo pathway. Inhibition of PKA and overexpression of YAP cooperate to transform normal cells and amplify neural progenitor pools in developing chick embryos. We also implicate neurofibromin 2 as an AKAP (A-kinase-anchoring protein) scaffold protein that facilitates the function of the cAMP/PKA-LATS-YAP pathway. Our study thus incorporates PKA as novel component of the Hippo pathway.

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APC^CDH1 Targets MgcRacGAP for Destruction in the Late M Phase

Nishimura Koutarou, Oki Toshihiko, Kitaura Jiro, Kuninaka Shinji, Saya Hideyuki, Sakaue-Sawano Asako, Miyawaki Atsushi, Kitamura Toshio

PLoS One 8 ( 5 ) 2013.05

Background:Male germ cell RacGTPase activating protein (MgcRacGAP) is an important regulator of the Rho family GTPases - RhoA, Rac1, and Cdc42 - and is indispensable in cytokinesis and cell cycle progression. Inactivation of RhoA by phosphorylated MgcRacGAP is an essential step in cytokinesis. MgcRacGAP is also involved in G1-S transition and nuclear transport of signal transducer and activator of transcription 3/5 (STAT3/5). Expression of MgcRacGAP is strictly controlled in a cell cycle-dependent manner. However, the underlying mechanisms have not been elucidated.Methodology/Principal Findings:Using MgcRacGAP deletion mutants and the fusion proteins of full-length or partial fragments of MgcRacGAP to mVenus fluorescent protein, we demonstrated that MgcRacGAP is degraded by the ubiquitin-proteasome pathway in the late M to G1 phase via APC^CDH1. We also identified the critical region for destruction located in the C-terminus of MgcRacGAP, AA537-570, which is necessary and sufficient for CDH1-mediated MgcRacGAP destruction. In addition, we identified a PEST domain-like structure with charged residues in MgcRacGAP and implicate it in effective ubiquitination of MgcRacGAP.Conclusions/Significance:Our findings not only reveal a novel mechanism for controlling the expression level of MgcRacGAP but also identify a new target of APC^CDH1. Moreover our results identify a C-terminal region AA537-570 of MgcRacGAP as its degron.

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The Arf/p53 protein module, which induces apoptosis, down-regulates histone H2AX to allow normal cells to survive in the presence of anti-cancer drugs

Atsumi Yuko, Inase Aki, Osawa Tomoyuki, Sugihara Eiji, Sakasai Ryo, Fujimori Hiroaki, Teraoka Hirobumi, Saya Hideyuki, Kanno Masamoto, Tashiro Fumio, Nakagama Hitoshi, Masutani Mitsuko, Yoshioka Ken Ichi

Journal of Biological Chemistry 288 ( 19 ) 13269 - 13277 2013.05

ISSN 0021-9258

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Correction

Muto Jun, Imai Takao, Ogawa Daisuke, Nishimoto Yoshinori, Okada Yohei, Mabuchi Yo, Kawase Takeshi, Iwanami Akio, Mischel Paul S., Saya Hideyuki, Yoshida Kazunari, Matsuzaki Yumi, Okano Hideyuki

PLoS One 8 ( 4 ) 2013.04

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