Faculty Profiles - Nakayama Masanori

Papers - Nakayama Masanori

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Examining the association between vaccine reactogenicity and antibody titer dynamics after the third dose of BNT162b2 vaccine using a mixed-effects model. International journal

Naomi Matsumoto, Hideharu Hagiya, Masanori Nakayama, Masanori Furukawa, Toshiharu Mitsuhashi, Soshi Takao, Fumio Otsuka, Takashi Yorifuji

Journal of infection and chemotherapy : official journal of the Japan Society of Chemotherapy 29 ( 1 ) 39 - 42 2023.1

Language：English Publishing type：Research paper (scientific journal)

BACKGROUND: To mitigate the COVID-19 pandemic, many countries have recommended the use of booster vaccinations. The relationship between the degree of adverse vaccine reactions and elevated antibody titers is of interest; however, no studies have investigated the temporal changes in antibody titers based on repeated measurements after a third dose of the BNT162b2 vaccine. METHODS: This prospective longitudinal cohort study was conducted with 62 healthcare workers who received a third dose of the BNT162b2 at Okayama University Hospital, Japan. Venous blood draw and fingertip whole blood test sample collection were conducted at the early (3-13 days) and 1-month time points; only FWT sample collection was conducted at the 2-month time point. Information on adverse reactions within 1 week after vaccination was also obtained. The association between fever of 37.5 °C or higher and antibody titers after the third dose of BNT162b2 was examined using a mixed-effects model and Poisson regression with robust variance. RESULTS: A trend toward higher antibody titers in the early period after vaccination was observed in the febrile individuals, but the differences were not significant at 1 and 2 months post-vaccination (the partial regression coefficient for fever was 8094.3 [-1910.2, 18,098.8] at 1 month after vaccination, and 1764.1 [-4133.9, 7662.1] at 2 months after vaccination in the adjusted models). CONCLUSION: The findings suggest that the presence of fever after the third vaccine does not predict a sustained elevation in serum antibody titers.

DOI： 10.1016/j.jiac.2022.09.012

Association Between Fever and Antibody Titer Trends After a Third Dose of the mRNA-1273 Vaccine.

Naomi Matsumoto, Tomoka Kadowaki, Rumi Matsuo, Ayako Sasaki, Chikara Miyaji, Chigusa Higuchi, Masanori Nakayama, Yasue Sakurada, Hideharu Hagiya, Soshi Takao, Fumio Otsuka, Takashi Yorifuji

Journal of epidemiology 32 ( 12 ) 567 - 569 2022.12

Early-stage antibody kinetics after the third dose of BNT162b2 mRNA COVID-19 vaccination measured by a point-of-care fingertip whole blood testing. International journal

Hideharu Hagiya, Yasuhiro Nakano, Masanori Furukawa, Naruhiko Sunada, Toru Hasegawa, Yasue Sakurada, Kou Hasegawa, Koichiro Yamamoto, Hiroko Ogawa, Takafumi Obara, Kouhei Ageta, Naomi Matsumoto, Rumi Matsuo, Tomoka Kadowaki, Akihito Higashikage, Takao Hikita, Takashi Yorifuji, Shinichi Toyooka, Yoshinobu Maeda, Yoshinori Yokokura, Fumio Otsuka, Masanori Nakayama

Scientific reports 12 ( 1 ) 20628 - 20628 2022.11

Language：English Publishing type：Research paper (scientific journal)

Amid the Coronavirus Disease 2019 pandemic, we aimed to demonstrate the accuracy of the fingertip whole blood sampling test (FWT) in measuring the antibody titer and uncovering its dynamics shortly after booster vaccination. Mokobio SARS-CoV-2 IgM & IgG Quantum Dot immunoassay (Mokobio Biotechnology R&D Center Inc., MD, USA) was used as a point-of-care FWT in 226 health care workers (HCWs) who had received two doses of the BNT162b2 mRNA vaccine (Pfizer-BioNTech) at least 8 months prior. Each participant tested their antibody titers before and after the third-dose booster up to 14-days. The effect of the booster was observed as early as the fourth day after vaccination, which exceeded the detection limit (> 30,000 U/mL) by 2.3% on the fifth day, 12.2% on the sixth day, and 22.5% after the seventh day. Significant positive correlations were observed between the pre- and post-vaccination (the seventh and eighth days) antibody titers (correlation coefficient, 0.405; p < 0.001). FWT is useful for examining antibody titers as a point-of-care test. Rapid response of antibody titer started as early as the fourth day post-vaccination, while the presence of weak responders to BNT162b2 vaccine was indicated.

DOI： 10.1038/s41598-022-24464-3

Poor vaccine responsiveness towards third-dose mRNA vaccine of COVID-19 in Japanese older people. International journal

Hideharu Hagiya, Takao Hikita, Tomohiro Habu, Masaki Asada, Takashi Yorifuji, Shinichi Toyooka, Fumio Otsuka, Masanori Nakayama

The Journal of infection 85 ( 4 ) 436 - 480 2022.10

Vasohibin-1が誘導する微小管の翻訳後修飾を介した受容体エンドサイトーシス阻害と新規脈管新生抑制機構

小林美穂, 若林育海, 藤原花汐, 鈴木康弘, 中山雅敬, 佐藤靖史, 渡部徹郎

リンパ学 44 ( 2 ) 65 - 69 2021.12

Numerical evaluation reveals the effect of branching morphology on vessel transport properties during angiogenesis. International journal

Fatemeh Mirzapour-Shafiyi, Yukinori Kametani, Takao Hikita, Yosuke Hasegawa, Masanori Nakayama

PLoS computational biology 17 ( 6 ) e1008398 2021.6

Language：English Publishing type：Research paper (scientific journal)

Blood flow governs transport of oxygen and nutrients into tissues. Hypoxic tissues secrete VEGFs to promote angiogenesis during development and in tissue homeostasis. In contrast, tumors enhance pathologic angiogenesis during growth and metastasis, suggesting suppression of tumor angiogenesis could limit tumor growth. In line with these observations, various factors have been identified to control vessel formation in the last decades. However, their impacts on the vascular transport properties of oxygen remain elusive. Here, we take a computational approach to examine the effects of vascular branching on blood flow in the growing vasculature. First of all, we reconstruct a 3D vascular model from the 2D confocal images of the growing vasculature at postnatal day 5 (P5) mouse retina, then simulate blood flow in the vasculatures, which are obtained from the gene targeting mouse models causing hypo- or hyper-branching vascular formation. Interestingly, hyper-branching morphology attenuates effective blood flow at the angiogenic front, likely promoting tissue hypoxia. In contrast, vascular hypo-branching enhances blood supply at the angiogenic front of the growing vasculature. Oxygen supply by newly formed blood vessels improves local hypoxia and decreases VEGF expression at the angiogenic front during angiogenesis. Consistent with the simulation results indicating improved blood flow in the hypo-branching vasculature, VEGF expression around the angiogenic front is reduced in those mouse retinas. Conversely, VEGF expression is enhanced in the angiogenic front of hyper-branching vasculature. Our results indicate the importance of detailed flow analysis in evaluating the vascular transport properties of branching morphology of the blood vessels.

DOI： 10.1371/journal.pcbi.1008398

Tubulin carboxypeptidase activity of vasohibin-1 inhibits angiogenesis by interfering with endocytosis and trafficking of pro-angiogenic factor receptors. Reviewed International journal

Miho Kobayashi, Ikumi Wakabayashi, Yasuhiro Suzuki, Kashio Fujiwara, Masanori Nakayama, Tetsuro Watabe, Yasufumi Sato

Angiogenesis 24 ( 1 ) 159 - 176 2020.10

Language：English Publishing type：Research paper (scientific journal)

Receptor endocytosis is crucial for integrating extracellular stimuli of pro-angiogenic factors, including vascular endothelial growth factor (VEGF), into the cell via signal transduction. VEGF not only triggers various angiogenic events including endothelial cell (EC) migration, but also induces the expression of negative regulators of angiogenesis, including vasohibin-1 (VASH1). While we have previously reported that VASH1 inhibits angiogenesis in vitro and in vivo, its mode of action on EC behavior remains elusive. Recently VASH1 was shown to have tubulin carboxypeptidase (TCP) activity, mediating the post-translational modification of microtubules (MTs) by detyrosination of α-tubulin within cells. However, the role of VASH1 TCP activity in angiogenesis has not yet been clarified. Here, we showed that VASH1 detyrosinated α-tubulin in ECs and suppressed in vitro and in vivo angiogenesis. In cultured ECs, VASH1 impaired endocytosis and trafficking of VEGF receptor 2 (VEGFR2), which resulted in the decreased signal transduction and EC migration. These effects of VASH1 could be restored by tubulin tyrosine ligase (TTL) in ECs, suggesting that detyrosination of α-tubulin negatively regulates angiogenesis. Furthermore, we found that detyrosinated tubulin-rich MTs were not adequate as trafficking rails for VEGFR2 endocytosis. Consistent with these results, inhibition of TCP activity of VASH1 led to the inhibition of VASH1-mediated suppression of VEGF-induced signals, EC migration, and in vivo angiogenesis. Our results indicate a novel mechanism of VASH1-mediated inhibition of pro-angiogenic factor receptor trafficking via modification of MTs.

DOI： 10.1007/s10456-020-09754-6

The mitochondrial outer membrane protein SYNJ2BP interacts with the cell adhesion molecule TMIGD1 and can recruit it to mitochondria. International journal

Christian Hartmann, Ysabel Alessa Schwietzer, Daniel Kummer, Nils Kirschnick, Esther Hoppe, Eva-Maria Thüring, Mark Glaesner-Ebnet, Frauke Brinkmann, Volker Gerke, Stefan Reuter, Masanori Nakayama, Klaus Ebnet

BMC molecular and cell biology 21 ( 1 ) 30 - 30 2020.4

Language：English Publishing type：Research paper (scientific journal)

BACKGROUND: Transmembrane and immunoglobulin domain-containing protein 1 (TMIGD1) is a recently identified cell adhesion molecule which is predominantly expressed by epithelial cells of the intestine and the kidney. Its expression is downregulated in both colon and renal cancer suggesting a tumor suppressive activity. The function of TMIGD1 at the cellular level is largely unclear. Published work suggests a protective role of TMIGD1 during oxidative stress in kidney epithelial cells, but the underlying molecular mechanisms are unknown. RESULTS: In this study, we address the subcellular localization of TMIGD1 in renal epithelial cells and identify a cytoplasmic scaffold protein as interaction partner of TMIGD1. We find that TMIGD1 localizes to different compartments in renal epithelial cells and that this localization is regulated by cell confluency. Whereas it localizes to mitochondria in subconfluent cells it is localized at cell-cell contacts in confluent cells. We find that cell-cell contact localization is regulated by N-glycosylation and that both the extracellular and the cytoplasmic domain contribute to this localization. We identify Synaptojanin 2-binding protein (SYNJ2BP), a PDZ domain-containing cytoplasmic protein, which localizes to both mitochondria and the plasma membrane, as interaction partner of TMIGD1. The interaction of TMIGD1 and SYNJ2BP is mediated by the PDZ domain of SYNJ2BP and the C-terminal PDZ domain-binding motif of TMIGD1. We also find that SYNJ2BP can actively recruit TMIGD1 to mitochondria providing a potential mechanism for the localization of TMIGD1 at mitochondria. CONCLUSIONS: This study describes TMIGD1 as an adhesion receptor that can localize to both mitochondria and cell-cell junctions in renal epithelial cells. It identifies SYNJ2BP as an interaction partner of TMIGD1 providing a potential mechanism underlying the localization of TMIGD1 at mitochondria. The study thus lays the basis for a better understanding of the molecular function of TMIGD1 during oxidative stress regulation.

DOI： 10.1186/s12860-020-00274-1

Does Abatacept Increase Postoperative Adverse Events in Rheumatoid Arthritis Compared with Conventional Synthetic Disease-modifying Drugs? International journal

Hiromu Ito, Shigeyoshi Tsuji, Masanori Nakayama, Yuichi Mochida, Keiichiro Nishida, Hajime Ishikawa, Toshihisa Kojima, Takumi Matsumoto, Ayako Kubota, Takeshi Mochizuki, Koji Sakuraba, Isao Matsushita, Arata Nakajima, Ryota Hara, Akihisa Haraguchi, Tsukasa Matsubara, Katsuaki Kanbe, Natsuko Nakagawa, Masahide Hamaguchi, Shigeki Momohara

The Journal of rheumatology 47 ( 4 ) 502 - 509 2020.4

Language：English Publishing type：Research paper (scientific journal)

OBJECTIVE: To investigate whether abatacept (ABA) causes more adverse events (AE) than conventional synthetic disease-modifying antirheumatic drugs (csDMARD) after orthopedic surgery in patients with rheumatoid arthritis (RA). METHODS: A retrospective multicenter nested case-control study was performed in 18 institutions. Patients receiving ABA (ABA group) were matched individually with patients receiving csDMARD and/or steroids (control group). Postoperative AE included surgical site infection, delayed wound healing, deep vein thrombosis or pulmonary embolism, flare, and death. The incidence rates of the AE in both groups were compared with the Mantel-Haenszel test. Risk factors for AE were analyzed by logistic regression model. RESULTS: A total of 3358 cases were collected. After inclusion and exclusion, 2651 patients were selected for matching, and 194 patients in 97 pairs were chosen for subsequent comparative analyses between the ABA and control groups. No between-group differences were detected in the incidence rates of each AE or in the incidence rates of total AE (control vs ABA: 15.5% vs 20.7% in total, 5.2% vs 3.1% in death). CONCLUSION: Compared with csDMARD and/or steroids without ABA, adding ABA to the treatment does not appear to increase the incidence rates of postoperative AE in patients with RA undergoing orthopedic surgery. Large cohort studies should be performed to add evidence for the perioperative safety profile of ABA.

DOI： 10.3899/jrheum.181100

Digenic inheritance of mutations in EPHA2 and SLC26A4 in Pendred syndrome. Reviewed International journal

Mengnan Li, Shin-Ya Nishio, Chie Naruse, Meghan Riddell, Sabrina Sapski, Tatsuya Katsuno, Takao Hikita, Fatemeh Mizapourshafiyi, Fiona M Smith, Leanne T Cooper, Min Goo Lee, Masahide Asano, Thomas Boettger, Marcus Krueger, Astrid Wietelmann, Johannes Graumann, Bryan W Day, Andrew W Boyd, Stefan Offermanns, Shin-Ichiro Kitajiri, Shin-Ichi Usami, Masanori Nakayama

Nature communications 11 ( 1 ) 1343 - 1343 2020.3

Language：English Publishing type：Research paper (scientific journal)

Enlarged vestibular aqueduct (EVA) is one of the most commonly identified inner ear malformations in hearing loss patients including Pendred syndrome. While biallelic mutations of the SLC26A4 gene, encoding pendrin, causes non-syndromic hearing loss with EVA or Pendred syndrome, a considerable number of patients appear to carry mono-allelic mutation. This suggests faulty pendrin regulatory machinery results in hearing loss. Here we identify EPHA2 as another causative gene of Pendred syndrome with SLC26A4. EphA2 forms a protein complex with pendrin controlling pendrin localization, which is disrupted in some pathogenic forms of pendrin. Moreover, point mutations leading to amino acid substitution in the EPHA2 gene are identified from patients bearing mono-allelic mutation of SLC26A4. Ephrin-B2 binds to EphA2 triggering internalization with pendrin inducing EphA2 autophosphorylation weakly. The identified EphA2 mutants attenuate ephrin-B2- but not ephrin-A1-induced EphA2 internalization with pendrin. Our results uncover an unexpected role of the Eph/ephrin system in epithelial function.

DOI： 10.1038/s41467-020-15198-9

Regulation of programmed cell death ligand 1 expression by atypical protein kinase C lambda/iota in cutaneous angiosarcoma. Reviewed International journal

Ai Kawamura, Takuji Kawamura, Meghan Riddell, Takao Hikita, Teruki Yanagi, Hiroshi Umemura, Masanori Nakayama

Cancer science 110 ( 5 ) 1780 - 1789 2019.5

Language：English Publishing type：Research paper (scientific journal)

The expression of immune checkpoint proteins such as programmed cell death protein 1 (PD-1) and its ligand (PD-L1) has been shown to correlate with patient prognosis in many malignant cancers. The expression of PD-L1 is controlled by c-Myc; however, further upstream regulation of PD-L1 expression is largely unknown. We have previously shown that atypical protein kinase C lambda/iota (aPKCλ) phosphorylates the Forkhead box protein O1 (FoxO1) transcription factor at Ser218 to suppress its DNA-binding ability, thereby regulating c-Myc expression and controlling physiologic and pathologic endothelial proliferation. The presence of phosphorylation of FoxO1 at Ser218 (pSer218 FoxO1) in cutaneous angiosarcoma (CAS) strongly correlates with poor patient prognosis. Here, we reported that patients with PD-L1+ cells in CAS lesions showed significantly worse prognosis compared to those that were PD-L1- . Expression of PD-L1 correlated with that of aPKCλ or the presence of pSer218FoxO1. Moreover, suppression of aPKCλ expression or inhibition of its activity in HUVECs or AS-M, an established human angiosarcoma cell line, resulted in decreased PD-L1 expression. Our results suggest that combined treatment with immune checkpoint inhibitors and aPKCλ inhibitors could be a novel treatment strategy for CAS patients.

DOI： 10.1111/cas.13981

aPKC controls endothelial growth by modulating c-Myc via FoxO1 DNA-binding ability. Reviewed International journal

Meghan Riddell, Akiko Nakayama, Takao Hikita, Fatemeh Mirzapourshafiyi, Takuji Kawamura, Ayesha Pasha, Mengnan Li, Mikio Masuzawa, Mario Looso, Tim Steinbacher, Klaus Ebnet, Michael Potente, Tomonori Hirose, Shigeo Ohno, Ingrid Fleming, Stefan Gattenlöhner, Phyu P Aung, Thuy Phung, Osamu Yamasaki, Teruki Yanagi, Hiroshi Umemura, Masanori Nakayama

Nature communications 9 ( 1 ) 5357 - 5357 2018.12

Language：English Publishing type：Research paper (scientific journal)

Strict regulation of proliferation is vital for development, whereas unregulated cell proliferation is a fundamental characteristic of cancer. The polarity protein atypical protein kinase C lambda/iota (aPKCλ) is associated with cell proliferation through unknown mechanisms. In endothelial cells, suppression of aPKCλ impairs proliferation despite hyperactivated mitogenic signaling. Here we show that aPKCλ phosphorylates the DNA binding domain of forkhead box O1 (FoxO1) transcription factor, a gatekeeper of endothelial growth. Although mitogenic signaling excludes FoxO1 from the nucleus, consequently increasing c-Myc abundance and proliferation, aPKCλ controls c-Myc expression via FoxO1/miR-34c signaling without affecting its localization. We find this pathway is strongly activated in the malignant vascular sarcoma, angiosarcoma, and aPKC inhibition reduces c-Myc expression and proliferation of angiosarcoma cells. Moreover, FoxO1 phosphorylation at Ser218 and aPKC expression correlates with poor patient prognosis. Our findings may provide a potential therapeutic strategy for treatment of malignant cancers, like angiosarcoma.

DOI： 10.1038/s41467-018-07739-0

PAR-3 controls endothelial planar polarity and vascular inflammation under laminar flow. Reviewed International journal

Takao Hikita, Fatemeh Mirzapourshafiyi, Pedro Barbacena, Meghan Riddell, Ayesha Pasha, Mengnan Li, Takuji Kawamura, Ralf P Brandes, Tomonori Hirose, Shigeo Ohno, Holger Gerhardt, Michiyuki Matsuda, Claudio A Franco, Masanori Nakayama

EMBO reports 19 ( 9 ) 2018.9

Language：English Publishing type：Research paper (scientific journal)

Impaired cell polarity is a hallmark of diseased tissue. In the cardiovascular system, laminar blood flow induces endothelial planar cell polarity, represented by elongated cell shape and asymmetric distribution of intracellular organelles along the axis of blood flow. Disrupted endothelial planar polarity is considered to be pro-inflammatory, suggesting that the establishment of endothelial polarity elicits an anti-inflammatory response. However, a causative relationship between polarity and inflammatory responses has not been firmly established. Here, we find that a cell polarity protein, PAR-3, is an essential gatekeeper of GSK3β activity in response to laminar blood flow. We show that flow-induced spatial distribution of PAR-3/aPKCλ and aPKCλ/GSK3β complexes controls local GSK3β activity and thereby regulates endothelial planar polarity. The spatial information for GSK3β activation is essential for flow-dependent polarity to the flow axis, but is not necessary for flow-induced anti-inflammatory response. Our results shed light on a novel relationship between endothelial polarity and vascular homeostasis highlighting avenues for novel therapeutic strategies.

DOI： 10.15252/embr.201745253

Regulation of cell polarity by cell adhesion receptors. International journal

Klaus Ebnet, Daniel Kummer, Tim Steinbacher, Amrita Singh, Masanori Nakayama, Maja Matis

Seminars in cell & developmental biology 81 2 - 12 2018.9

Language：English Publishing type：Research paper (scientific journal)

The ability of cells to polarize is an intrinsic property of almost all cells and is required for the devlopment of most multicellular organisms. To develop cell polarity, cells integrate various signals derived from intrinsic as well as extrinsic sources. In the recent years, cell-cell adhesion receptors have turned out as important regulators of cellular polarization. By interacting with conserved cell polarity proteins, they regulate the recruitment of polarity complexes to specific sites of cell-cell adhesion. By initiating intracellular signaling cascades at those sites, they trigger their specific subcellular activation. Not surprisingly, cell-cell adhesion receptors regulate diverse aspects of cell polarity, including apico-basal polarity in epithelial and endothelial cells, front-to-rear polarity in collectively migrating cells, and planar cell polarity during organ development. Here, we review the recent developments highlighting the central roles of cell-cell adhesion molecules in the development of cell polarity.

DOI： 10.1016/j.semcdb.2017.07.032

Pericytes regulate VEGF-induced endothelial sprouting through VEGFR1. Reviewed International journal

Hanna M Eilken, Rodrigo Diéguez-Hurtado, Inga Schmidt, Masanori Nakayama, Hyun-Woo Jeong, Hendrik Arf, Susanne Adams, Napoleone Ferrara, Ralf H Adams

Nature communications 8 ( 1 ) 1574 - 1574 2017.11

Language：English Publishing type：Research paper (scientific journal)

Pericytes adhere to the abluminal surface of endothelial tubules and are required for the formation of stable vascular networks. Defective endothelial cell-pericyte interactions are frequently observed in diseases characterized by compromised vascular integrity such as diabetic retinopathy. Many functional properties of pericytes and their exact role in the regulation of angiogenic blood vessel growth remain elusive. Here we show that pericytes promote endothelial sprouting in the postnatal retinal vasculature. Using genetic and pharmacological approaches, we show that the expression of vascular endothelial growth factor receptor 1 (VEGFR1) by pericytes spatially restricts VEGF signalling. Angiogenic defects caused by pericyte depletion are phenocopied by intraocular injection of VEGF-A or pericyte-specific inactivation of the murine gene encoding VEGFR1. Our findings establish that pericytes promote endothelial sprouting, which results in the loss of side branches and the enlargement of vessels when pericyte function is impaired or lost.

DOI： 10.1038/s41467-017-01738-3

VE-cadherin interacts with cell polarity protein Pals1 to regulate vascular lumen formation. International journal

Benjamin F Brinkmann, Tim Steinbacher, Christian Hartmann, Daniel Kummer, Denise Pajonczyk, Fatemeh Mirzapourshafiyi, Masanori Nakayama, Thomas Weide, Volker Gerke, Klaus Ebnet

Molecular biology of the cell 27 ( 18 ) 2811 - 21 2016.9

Language：English Publishing type：Research paper (scientific journal)

Blood vessel tubulogenesis requires the formation of stable cell-to-cell contacts and the establishment of apicobasal polarity of vascular endothelial cells. Cell polarity is regulated by highly conserved cell polarity protein complexes such as the Par3-aPKC-Par6 complex and the CRB3-Pals1-PATJ complex, which are expressed by many different cell types and regulate various aspects of cell polarity. Here we describe a functional interaction of VE-cadherin with the cell polarity protein Pals1. Pals1 directly interacts with VE-cadherin through a membrane-proximal motif in the cytoplasmic domain of VE-cadherin. VE-cadherin clusters Pals1 at cell-cell junctions. Mutating the Pals1-binding motif in VE-cadherin abrogates the ability of VE-cadherin to regulate apicobasal polarity and vascular lumen formation. In a similar way, deletion of the Par3-binding motif at the C-terminus of VE-cadherin impairs apicobasal polarity and vascular lumen formation. Our findings indicate that the biological activity of VE-cadherin in regulating endothelial polarity and vascular lumen formation is mediated through its interaction with the two cell polarity proteins Pals1 and Par3.

DOI： 10.1091/mbc.E16-02-0127

Regulation of vascular endothelial growth factor receptor function in angiogenesis by numb and numb-like. Reviewed International journal

Max van Lessen, Masanori Nakayama, Katsuhiro Kato, Jung Mo Kim, Kozo Kaibuchi, Ralf H Adams

Arteriosclerosis, thrombosis, and vascular biology 35 ( 8 ) 1815 - 25 2015.8

Language：English Publishing type：Research paper (scientific journal)

OBJECTIVE: Vascular endothelial growth factor (VEGF) signaling is a major regulator of physiological and pathological angiogenesis. VEGF receptor activity is strongly controlled by endocytosis, which can terminate or enhance signal transduction in the angiogenic endothelium, but the exact molecular regulation of these processes remains incompletely understood. We have therefore examined the function of Numb family clathrin-associated sorting proteins in angiogenesis. APPROACH AND RESULTS: We show that Numb proteins are expressed by endothelial cells during retinal angiogenesis in mice. Inducible inactivation of the Numb/Numbl genes in the postnatal endothelium led to impaired vessel growth, reduced endothelial proliferation and sprouting, and decreased VEGF receptor activation. Biochemistry and cell biology experiments established that Numb can interact with VEGFR2 and VEGFR3 and controls VEGF receptor activation in response to ligand stimulation. Experiments in cultured endothelial cells showed that Numb proteins counteract VEGF receptor degradation and promote VEGFR2 recycling back to the plasma membrane. CONCLUSIONS: Numb proteins control VEGF receptor endocytosis, signaling, and recycling in endothelial cells, which promotes the angiogenic growth of blood vessels.

DOI： 10.1161/ATVBAHA.115.305473

Integrin β1 controls VE-cadherin localization and blood vessel stability. Reviewed International journal

Hiroyuki Yamamoto, Manuel Ehling, Katsuhiro Kato, Kenichi Kanai, Max van Lessen, Maike Frye, Dagmar Zeuschner, Masanori Nakayama, Dietmar Vestweber, Ralf H Adams

Nature communications 6 6429 - 6429 2015.3

Language：English Publishing type：Research paper (scientific journal)

Angiogenic blood vessel growth requires several distinct but integrated cellular activities. Endothelial cell sprouting and proliferation lead to the expansion of the vasculature and give rise to a highly branched, immature plexus, which is subsequently reorganized into a mature and stable network. Although it is known that integrin-mediated cell-matrix interactions are indispensable for embryonic angiogenesis, little is known about the function of integrins in different steps of vascular morphogenesis. Here, by investigating the integrin β1-subunit with inducible and endothelial-specific gene targeting in the postnatal mouse retina, we show that β1 integrin promotes endothelial sprouting but is a negative regulator of proliferation. In maturing vessels, integrin β1 is indispensable for proper localization of VE-cadherin and thereby cell-cell junction integrity. The sum of our findings establishes that integrin β1 has critical functions in the growing and maturing vasculature, and is required for the formation of stable, non-leaky blood vessels.

DOI： 10.1038/ncomms7429

Ephrin-B2 controls PDGFRβ internalization and signaling. Reviewed International journal

Akiko Nakayama, Masanori Nakayama, Christopher J Turner, Susanne Höing, John J Lepore, Ralf H Adams

Genes & development 27 ( 23 ) 2576 - 89 2013.12

Language：English Publishing type：Research paper (scientific journal)

B-class ephrins, ligands for EphB receptor tyrosine kinases, are critical regulators of growth and patterning processes in many organs and species. In the endothelium of the developing vasculature, ephrin-B2 controls endothelial sprouting and proliferation, which has been linked to vascular endothelial growth factor (VEGF) receptor endocytosis and signaling. Ephrin-B2 also has essential roles in supporting mural cells (namely, pericytes and vascular smooth muscle cells [VSMCs]), but the underlying mechanism is not understood. Here, we show that ephrin-B2 controls platelet-derived growth factor receptor β (PDGFRβ) distribution in the VSMC plasma membrane, endocytosis, and signaling in a fashion that is highly distinct from its role in the endothelium. Absence of ephrin-B2 in cultured VSMCs led to the redistribution of PDGFRβ from caveolin-positive to clathrin-associated membrane fractions, enhanced PDGF-B-induced PDGFRβ internalization, and augmented downstream mitogen-activated protein (MAP) kinase and c-Jun N-terminal kinase (JNK) activation but impaired Tiam1-Rac1 signaling and proliferation. Accordingly, mutant mice lacking ephrin-B2 expression in vascular smooth muscle developed vessel wall defects and aortic aneurysms, which were associated with impaired Tiam1 expression and excessive activation of MAP kinase and JNK. Our results establish that ephrin-B2 is an important regulator of PDGFRβ endocytosis and thereby acts as a molecular switch controlling the downstream signaling activity of this receptor in mural cells.

DOI： 10.1101/gad.224089.113

Coordination of VEGF receptor trafficking and signaling by coreceptors. Reviewed International journal

Masanori Nakayama, Philipp Berger

Experimental cell research 319 ( 9 ) 1340 - 7 2013.5

Language：English Publishing type：Research paper (scientific journal)

During development, regeneration and in certain pathological settings, the vasculature is expanded and remodeled substantially. Proper morphogenesis and function of blood vessels are essential in multicellular organisms. Upon stimulation with growth factors including vascular endothelial growth factors (VEGFs), the activation, internalization and sorting of receptor tyrosine kinases (RTKs) orchestrate developmental processes and the homeostatic maintenance of all organs including the vasculature. Previously, RTK signaling was thought to occur exclusively at the plasma membrane, a process that was subsequently terminated by endocytosis and receptor degradation. However, this model turned out to be an oversimplification and there is now a substantial amount of reports indicating that receptor internalization and trafficking to intracellular compartments depends on coreceptors leading to the activation of specific signaling pathways. Here we review the latest findings concerning endocytosis and intracellular trafficking of VEGFRs. The body of evidence is compelling that VEGF receptor trafficking is coordinated with other proteins such as Neuropilin-1, ephrin-B2, VE-cadherin and protein phosphatases.

DOI： 10.1016/j.yexcr.2013.03.008

Tetraspanin CD9 links junctional adhesion molecule-A to αvβ3 integrin to mediate basic fibroblast growth factor-specific angiogenic signaling. Reviewed International journal

Swetha S D Peddibhotla, Benjamin F Brinkmann, Daniel Kummer, Hüseyin Tuncay, Masanori Nakayama, Ralf H Adams, Volker Gerke, Klaus Ebnet

Molecular biology of the cell 24 ( 7 ) 933 - 44 2013.4

Language：English Publishing type：Research paper (scientific journal)

Junctional adhesion molecule-A (JAM-A) is a member of the immunoglobulin family with diverse functions in epithelial cells, including cell migration, cell contact maturation, and tight junction formation. In endothelial cells, JAM-A has been implicated in basic fibroblast growth factor (bFGF)-regulated angiogenesis through incompletely understood mechanisms. In this paper, we identify tetraspanin CD9 as novel binding partner for JAM-A in endothelial cells. CD9 acts as scaffold and assembles a ternary JAM-A-CD9-αvβ3 integrin complex from which JAM-A is released upon bFGF stimulation. CD9 interacts predominantly with monomeric JAM-A, which suggests that bFGF induces signaling by triggering JAM-A dimerization. Among the two vitronectin receptors, αvβ3 and αvβ5 integrin, which have been shown to cooperate during angiogenic signaling with bFGF and vascular endothelial growth factor (VEGF), respectively, CD9 links JAM-A specifically to αvβ3 integrin. In line with this, knockdown of CD9 blocks bFGF- but not VEGF-induced ERK1/2 activation. JAM-A or CD9 knockdown impairs endothelial cell migration and tube formation. Our findings indicate that CD9 incorporates monomeric JAM-A into a complex with αvβ3 integrin, which responds to bFGF stimulation by JAM-A release to regulate mitogen-activated protein kinase (MAPK) activation, endothelial cell migration, and angiogenesis. The data also provide new mechanistic insights into the cooperativity between bFGF and αvβ3 integrin during angiogenic signaling.

DOI： 10.1091/mbc.E12-06-0481

Spatial regulation of VEGF receptor endocytosis in angiogenesis. Reviewed International journal

Masanori Nakayama, Akiko Nakayama, Max van Lessen, Hiroyuki Yamamoto, Sarah Hoffmann, Hannes C A Drexler, Norimichi Itoh, Tomonori Hirose, Georg Breier, Dietmar Vestweber, Jonathan A Cooper, Shigeo Ohno, Kozo Kaibuchi, Ralf H Adams

Nature cell biology 15 ( 3 ) 249 - 60 2013.3

Language：English Publishing type：Research paper (scientific journal)

Activities as diverse as migration, proliferation and patterning occur simultaneously and in a coordinated fashion during tissue morphogenesis. In the growing vasculature, the formation of motile, invasive and filopodia-carrying endothelial sprouts is balanced with the stabilization of blood-transporting vessels. Here, we show that sprouting endothelial cells in the retina have high rates of VEGF uptake, VEGF receptor endocytosis and turnover. These internalization processes are opposed by atypical protein kinase C activity in more stable and mature vessels. aPKC phosphorylates Dab2, a clathrin-associated sorting protein that, together with the transmembrane protein ephrin-B2 and the cell polarity regulator PAR-3, enables VEGF receptor endocytosis and downstream signal transduction. Accordingly, VEGF receptor internalization and the angiogenic growth of vascular beds are defective in loss-of-function mice lacking key components of this regulatory pathway. Our work uncovers how vessel growth is dynamically controlled by local VEGF receptor endocytosis and the activity of cell polarity proteins.

DOI： 10.1038/ncb2679

Proteomic screening for Rho-kinase substrates by combining kinase and phosphatase inhibitors with 14-3-3ζ affinity chromatography. Reviewed

Tomoki Nishioka, Masanori Nakayama, Mutsuki Amano, Kozo Kaibuchi

Cell structure and function 37 ( 1 ) 39 - 48 2012

Language：English Publishing type：Research paper (scientific journal)

The small GTPase RhoA is a molecular switch in various extracellular signals. Rho-kinase/ROCK/ROK, a major effector of RhoA, regulates diverse cellular functions by phosphorylating cytoskeletal proteins, endocytic proteins, and polarity proteins. More than twenty Rho-kinase substrates have been reported, but the known substrates do not fully explain the Rho-kinase functions. Herein, we describe the comprehensive screening for Rho-kinase substrates by treating HeLa cells with Rho-kinase and phosphatase inhibitors. The cell lysates containing the phosphorylated substrates were then subjected to affinity chromatography using beads coated with 14-3-3 protein, which interacts with proteins containing phosphorylated serine or threonine residues, to enrich the phosphorylated proteins. The identities of the molecules and phosphorylation sites were determined by liquid chromatography tandem mass spectrometry (LC/MS/MS) after tryptic digestion and phosphopeptide enrichment. The phosphorylated proteins whose phosphopeptide ion peaks were suppressed by treatment with the Rho-kinase inhibitor were regarded as candidate substrates. We identified 121 proteins as candidate substrates. We also identified phosphorylation sites in Partitioning defective 3 homolog (Par-3) at Ser143 and Ser144. We found that Rho-kinase phosphorylated Par-3 at Ser144 both in vitro and in vivo. The method used in this study would be applicable and useful to identify novel substrates of other kinases.

Involvement of Girdin in the determination of cell polarity during cell migration. Reviewed International journal

Kei Ohara, Atsushi Enomoto, Takuya Kato, Takahiko Hashimoto, Mayu Isotani-Sakakibara, Naoya Asai, Maki Ishida-Takagishi, Liang Weng, Masanori Nakayama, Takashi Watanabe, Katsuhiro Kato, Kozo Kaibuchi, Yoshiki Murakumo, Yoshiki Hirooka, Hidemi Goto, Masahide Takahashi

PloS one 7 ( 5 ) e36681 2012

Language：English Publishing type：Research paper (scientific journal)

Cell migration is a critical cellular process that determines embryonic development and the progression of human diseases. Therefore, cell- or context-specific mechanisms by which multiple promigratory proteins differentially regulate cell migration must be analyzed in detail. Girdin (girders of actin filaments) (also termed GIV, Gα-interacting vesicle associated protein) is an actin-binding protein that regulates migration of various cells such as endothelial cells, smooth muscle cells, neuroblasts, and cancer cells. Here we show that Girdin regulates the establishment of cell polarity, the deregulation of which may result in the disruption of directional cell migration. We found that Girdin interacts with Par-3, a scaffolding protein that is a component of the Par protein complex that has an established role in determining cell polarity. RNA interference-mediated depletion of Girdin leads to impaired polarization of fibroblasts and mammary epithelial cells in a way similar to that observed in Par-3-depleted cells. Accordingly, the expression of Par-3 mutants unable to interact with Girdin abrogates cell polarization in fibroblasts. Further biochemical analysis suggests that Girdin is present in the Par protein complex that includes Par-3, Par-6, and atypical protein kinase C. Considering previous reports showing the role of Girdin in the directional migration of neuroblasts, network formation of endothelial cells, and cancer invasion, these data may provide a specific mechanism by which Girdin regulates cell movement in biological contexts that require directional cell movement.

DOI： 10.1371/journal.pone.0036681

Screening of Rho kinase substrates by combining inhibitors of protein kinase and phosphatase

Nishioka Tomoki, Nakayama Masanori, Amano Mutsuki, Kaibuchi Kozo

Abstracts for Annual Meeting of Japanese Proteomics Society 2011 131 - 131 2011

EphB signaling directs peripheral nerve regeneration through Sox2-dependent Schwann cell sorting. Reviewed International journal

Simona Parrinello, Ilaria Napoli, Sara Ribeiro, Patrick Wingfield Digby, Marina Fedorova, David B Parkinson, Robin D S Doddrell, Masanori Nakayama, Ralf H Adams, Alison C Lloyd

Cell 143 ( 1 ) 145 - 55 2010.10

Language：English Publishing type：Research paper (scientific journal)

The peripheral nervous system has astonishing regenerative capabilities in that cut nerves are able to reconnect and re-establish their function. Schwann cells are important players in this process, during which they dedifferentiate to a progenitor/stem cell and promote axonal regrowth. Here, we report that fibroblasts also play a key role. Upon nerve cut, ephrin-B/EphB2 signaling between fibroblasts and Schwann cells results in cell sorting, followed by directional collective cell migration of Schwann cells out of the nerve stumps to guide regrowing axons across the wound. Mechanistically, we find that cell-sorting downstream of EphB2 is mediated by the stemness factor Sox2 through N-cadherin relocalization to Schwann cell-cell contacts. In vivo, loss of EphB2 signaling impaired organized migration of Schwann cells, resulting in misdirected axonal regrowth. Our results identify a link between Ephs and Sox proteins, providing a mechanism by which progenitor cells can translate environmental cues to orchestrate the formation of new tissue.

DOI： 10.1016/j.cell.2010.08.039

Rho-kinase/ROCK: A key regulator of the cytoskeleton and cell polarity. Reviewed International journal

Mutsuki Amano, Masanori Nakayama, Kozo Kaibuchi

Cytoskeleton (Hoboken, N.J.) 67 ( 9 ) 545 - 54 2010.9

Language：English Publishing type：Research paper (scientific journal)

Rho-associated kinase (Rho-kinase/ROCK/ROK) is an effector of the small GTPase Rho and belongs to the AGC family of kinases. Rho-kinase has pleiotropic functions including the regulation of cellular contraction, motility, morphology, polarity, cell division, and gene expression. Pharmacological analyses have revealed that Rho-kinase is involved in a wide range of diseases such as vasospasm, pulmonary hypertension, nerve injury, and glaucoma, and is therefore considered to be a potential therapeutic target. This review focuses on the structure, function, and modes of activation and action of Rho-kinase.

DOI： 10.1002/cm.20472

Ephrin-B2 controls VEGF-induced angiogenesis and lymphangiogenesis. Reviewed International journal

Yingdi Wang, Masanori Nakayama, Mara E Pitulescu, Tim S Schmidt, Magdalena L Bochenek, Akira Sakakibara, Susanne Adams, Alice Davy, Urban Deutsch, Urs Lüthi, Alcide Barberis, Laura E Benjamin, Taija Mäkinen, Catherine D Nobes, Ralf H Adams

Nature 465 ( 7297 ) 483 - 6 2010.5

Language：English Publishing type：Research paper (scientific journal)

In development, tissue regeneration or certain diseases, angiogenic growth leads to the expansion of blood vessels and the lymphatic vasculature. This involves endothelial cell proliferation as well as angiogenic sprouting, in which a subset of cells, termed tip cells, acquires motile, invasive behaviour and extends filopodial protrusions. Although it is already appreciated that angiogenesis is triggered by tissue-derived signals, such as vascular endothelial growth factor (VEGF) family growth factors, the resulting signalling processes in endothelial cells are only partly understood. Here we show with genetic experiments in mouse and zebrafish that ephrin-B2, a transmembrane ligand for Eph receptor tyrosine kinases, promotes sprouting behaviour and motility in the angiogenic endothelium. We link this pro-angiogenic function to a crucial role of ephrin-B2 in the VEGF signalling pathway, which we have studied in detail for VEGFR3, the receptor for VEGF-C. In the absence of ephrin-B2, the internalization of VEGFR3 in cultured cells and mutant mice is defective, which compromises downstream signal transduction by the small GTPase Rac1, Akt and the mitogen-activated protein kinase Erk. Our results show that full VEGFR3 signalling is coupled to receptor internalization. Ephrin-B2 is a key regulator of this process and thereby controls angiogenic and lymphangiogenic growth.

DOI： 10.1038/nature09002

Identification of focal adhesion kinase (FAK) and phosphatidylinositol 3-kinase (PI3-kinase) as Par3 partners by proteomic analysis. Reviewed International journal

Norimichi Itoh, Masanori Nakayama, Takashi Nishimura, Shin Fujisue, Tomoki Nishioka, Takashi Watanabe, Kozo Kaibuchi

Cytoskeleton (Hoboken, N.J.) 67 ( 5 ) 297 - 308 2010.5

Language：English Publishing type：Research paper (scientific journal)

Partition defective 3 (Par3) is involved in a variety of polarity events including establishment of apico-basal polarity of epithelial cell, axon/dendrite specification of neurons and directional migration of cells with front-rear polarity. Par3 is thought to regulate cell polarity as a scaffold protein by interacting with various partner proteins such as Par6, aPKC, Tiam1/2 and Numb. However, the mode of actions of Par3 in polarized migration remains largely unknown. To explore Par3 functions, we screened Par3-interacting proteins by combining Par3 affinity column chromatography and shotgun analysis using liquid chromatography-tandem mass spectrometry (LC-MS/MS). We obtained about two hundred Par3-interacting proteins from the rat brain cytosol fraction. Among them, we focused on FAK and PI3-kinase, as both of them participate in directional cell migration. FAK associated with the PDZ domain and the coiled-coil region of Par3 and p110 of PI3-kinase associated with the coiled-coil region of Par3. Par3 was partially colocalized with FAK in spreading cells. Depletion of Par3 by RNA interference inhibited adhesion-induced activation of FAK and PI3-kinase, and RNA interference-resistant Par3 restored the inhibitory effects. In addition, Par3 was required for the adhesion-induced cell spreading as well as for directional cell migration toward collagen. These results suggest that Par3 directly interacts with FAK and PI3-kinase, enhancing their activities for polarized cell migration.

DOI： 10.1002/cm.20444

ケラチン結合蛋白質アルバトロスは上皮細胞極性を制御する(The keratin-binding protein Albatross regulates polarization of epithelial cells)

猪子誠人, 杉本昌彦, 白水崇, 中山雅敬, 鄒鵬, 米村重信, 林裕子, 井澤一郎, 佐宗幹夫, 宇治幸隆, 貝淵弘三, 清野透, 稲垣昌樹

日本細胞生物学会大会講演要旨集 61回 176 - 176 2009.5

The keratin-binding protein Albatross regulates polarization of epithelial cells. Reviewed International journal

Masahiko Sugimoto, Akihito Inoko, Takashi Shiromizu, Masanori Nakayama, Peng Zou, Shigenobu Yonemura, Yuko Hayashi, Ichiro Izawa, Mikio Sasoh, Yukitaka Uji, Kozo Kaibuchi, Tohru Kiyono, Masaki Inagaki

The Journal of cell biology 183 ( 1 ) 19 - 28 2008.10

Language：English Publishing type：Research paper (scientific journal)

The keratin intermediate filament network is abundant in epithelial cells, but its function in the establishment and maintenance of cell polarity is unclear. Here, we show that Albatross complexes with Par3 to regulate formation of the apical junctional complex (AJC) and maintain lateral membrane identity. In nonpolarized epithelial cells, Albatross localizes with keratin filaments, whereas in polarized epithelial cells, Albatross is primarily localized in the vicinity of the AJC. Knockdown of Albatross in polarized cells causes a disappearance of key components of the AJC at cell-cell borders and keratin filament reorganization. Lateral proteins E-cadherin and desmoglein 2 were mislocalized even on the apical side. Although Albatross promotes localization of Par3 to the AJC, Par3 and ezrin are still retained at the apical surface in Albatross knockdown cells, which retain intact microvilli. Analysis of keratin-deficient epithelial cells revealed that keratins are required to stabilize the Albatross protein, thus promoting the formation of AJC. We propose that keratins and the keratin-binding protein Albatross are important for epithelial cell polarization.

DOI： 10.1083/jcb.200803133

ケラチン結合蛋白質Albatrossは上皮細胞の極性化を制御する(The keratin-binding protein Albatross regulates the polarization of epithelial cells)

猪子誠人, 杉本昌彦, 白水崇, 中山雅敬, 鄒鵬, 米村重信, 林裕子, 井澤一郎, 佐宗幹夫, 宇治幸隆, 貝淵弘三, 清野透, 稲垣昌樹

日本細胞生物学会大会講演要旨集 60回 123 - 123 2008.6

Rho-kinase phosphorylates PAR-3 and disrupts PAR complex formation. Reviewed International journal

Masanori Nakayama, Takaaki M Goto, Masayuki Sugimoto, Takashi Nishimura, Takafumi Shinagawa, Sigeo Ohno, Mutsuki Amano, Kozo Kaibuchi

Developmental cell 14 ( 2 ) 205 - 15 2008.2

Language：English Publishing type：Research paper (scientific journal)

A polarity complex of PAR-3, PAR-6, and atypical protein kinase C (aPKC) functions in various cell polarization events. PAR-3 directly interacts with Tiam1/Taim2 (STEF), Rac1-specific guanine nucleotide exchange factors, and forms a complex with aPKC-PAR-6-Cdc42*GTP, leading to Rac1 activation. RhoA antagonizes Rac1 in certain types of cells. However, the relationship between RhoA and the PAR complex remains elusive. We found here that Rho-kinase/ROCK/ROK, the effector of RhoA, phosphorylated PAR-3 at Thr833 and thereby disrupted its interaction with aPKC and PAR-6, but not with Tiam2. Phosphorylated PAR-3 was observed in the leading edge, and in central and rear portions of migrating cells having front-rear polarity. Knockdown of PAR-3 by small interfering RNA (siRNA) impaired cell migration, front-rear polarization, and PAR-3-mediated Rac1 activation, which were recovered with siRNA-resistant PAR-3, but not with the phospho-mimic PAR-3 mutant. We propose that RhoA/Rho-kinase inhibits PAR complex formation through PAR-3 phosphorylation, resulting in Rac1 inactivation.

DOI： 10.1016/j.devcel.2007.11.021

Rho-kinase phosphorylates eNOS at threonine 495 in endothelial cells. Reviewed International journal

Masayuki Sugimoto, Masanori Nakayama, Takaaki M Goto, Mutsuki Amano, Kimihiro Komori, Kozo Kaibuchi

Biochemical and biophysical research communications 361 ( 2 ) 462 - 7 2007.9

Language：English Publishing type：Research paper (scientific journal)

Endothelial nitric oxide synthase (eNOS) produces nitric oxide (NO), which is involved in various physiological functions of the cardiovascular system. eNOS is activated by dephosphorylation at Thr495 and phosphorylation at Ser1177. Inhibition of Rho-kinase, an effector of the small GTPase RhoA, leads to activation of Akt/PKB, which phosphorylates eNOS at Ser1177 and thereby promotes NO production. However, little is known about the effects of Rho-kinase on phosphorylation of Thr495. We here found that the constitutively active form of Rho-kinase phosphorylated eNOS at Thr495 in vitro. Expression of the constitutively active form of RhoA or Rho-kinase increased this phosphorylation in COS-7 cells. Addition of thrombin to cultured human umbilical vein endothelial cells induced phosphorylation of eNOS at Thr495. Treatment with Y27632, a Rho-kinase inhibitor, suppressed thrombin-induced phosphorylation at Thr495. These results indicate that Rho-kinase can directly phosphorylate eNOS at Thr495 to suppress NO production in endothelium.

Rho-kinase modulates the function of STEF, a Rac GEF, through its phosphorylation. Reviewed International journal

Mikito Takefuji, Kazutaka Mori, Yasuhiro Morita, Nariko Arimura, Takashi Nishimura, Masanori Nakayama, Mikio Hoshino, Akihiro Iwamatsu, Toyoaki Murohara, Kozo Kaibuchi, Mutsuki Amano

Biochemical and biophysical research communications 355 ( 3 ) 788 - 94 2007.4

Language：English Publishing type：Research paper (scientific journal)

Rho family GTPases are key regulators of various physiological processes. Several recent studies indicated that the antagonistic relationship between Rho and Rac is essential for cell polarity and that the Rac activity is negatively regulated by Rho. In this study, we found that Rho-kinase, an effector of Rho, counteracted the Rac GEF STEF-induced Rac1 activation in COS7 cells. Rho-kinase phosphorylated STEF at Thr1662 in vitro, and Y-27632, a Rho-kinase inhibitor, suppressed lysophosphatidic acid-induced phosphorylation of STEF in PC12D cells. STEF interacted with specific molecules such as microtubule-associated protein 1B, and the phosphorylation of STEF by Rho-kinase diminished its interaction with these molecules. STEF promoted nerve growth factor-induced neurite outgrowth in PC12D cells, while the phosphomimic mutant of STEF had a weakened ability to enhance neurite outgrowth. Taken together, these results suggest that the phosphorylation of STEF by Rho-kinase exerts the inhibitory effect on the function of STEF.

Hydrophilic statin suppresses vein graft intimal hyperplasia via endothelial cell-tropic Rho-kinase inhibition. Reviewed International journal

Dai Yamanouchi, Hiroshi Banno, Masanori Nakayama, Masayuki Sugimoto, Hiromine Fujita, Masayoshi Kobayashi, Hiroyuki Kuwano, Kimihiro Komori

Journal of vascular surgery 42 ( 4 ) 757 - 64 2005.10

Language：English Publishing type：Research paper (scientific journal)

BACKGROUND: Recent studies suggest that statins can protect the vasculature in a manner that is independent of their lipid-lowering activity through inhibition of the small guanosine triphosphate-binding protein, Rho, and Rho-associated kinase. Little information is available on the inhibitory effect of statins on vein graft intimal hyperplasia, the main cause of late graft failure after bypass grafting. We therefore examined the effects of a hydrophilic statin on vein graft intimal hyperplasia in vivo and Rho-kinase activity in vitro. METHODS: In the first experiment, rabbits were randomized to a control group (n = 7) that was fed regular rabbit chow or to a pravastatin group (n = 7) that was fed regular rabbit chow supplemented with 10 mg/kg pravastatin sodium. The branches of the jugular vein were ligated and an approximately 3-cm segment of the jugular vein was taken for an autologous reversed-vein graft. The carotid artery was cut and replaced with the harvested autologous jugular vein. At 2 and 4 weeks after the operation, vein grafts in both groups were harvested, and intimal hyperplasia of the vein grafts was assessed. In the second experiment, human umbilical vein endothelial cells and vascular smooth muscle cells were cultured and then treated with 1 micromol/L and 30 micromol/L pravastatin for 24 hours and harvested. Immunoblotting was performed on the resulting precipitates. Quantitative evaluation of phosphorylated myosin binding subunit and endothelial nitric oxide synthase was performed by densitometric analysis. RESULTS: We demonstrated that oral administration of the hydrophilic statin pravastatin to normocholesterolemic rabbits inhibited intimal hyperplasia of carotid interposition-reversed jugular vein grafts 4 weeks after implantation (pravastatin group, 39.5 +/- 3.5 microm vs control group, 64.0 +/- 7.1 microm; n = 7; P < .05) and suppressed cell proliferation and apoptosis in the neointima 2 weeks after implantation. In addition, we found that pravastatin inhibited Rho-kinase activity and accelerated endothelial nitric oxide synthase expression in human umbilical vein endothelial cells but did not inhibit Rho-kinase activity in vascular smooth muscle cells. CONCLUSIONS: These novel findings clearly demonstrate that a hydrophilic statin can suppress intimal hyperplasia of the vein graft in vivo and also show endothelial cell-tropic inhibition of Rho-kinase in vitro. Furthermore, these results strongly support the clinical use of hydrophilic statins to prevent intimal hyperplasia of the vein graft after bypass grafting. CLINICAL RELEVANCE: Late graft failure caused by neointimal hyperplasia limits the efficacy of vein grafting. Various treatments were examined to reduce neointimal hyperplasia, but a standard clinical treatment has not yet been established. We report here the inhibitory effect of pravastatin on the development of vein graft intimal hyperplasia. In addition, we demonstrate that pravastatin showed endothelial cell-tropic benefits through both the inhibition of Rho-kinase activity and acceleration of eNOS expression in vitro. Because the clinical benefits and safety of pravastatin have been established to a certain extent through long-term clinical usage, pravastatin may soon become standard treatment after vein bypass grafting.

Rho mediates endocytosis of epidermal growth factor receptor through phosphorylation of endophilin A1 by Rho-kinase. Reviewed International journal

Takako Kaneko, Akio Maeda, Mikito Takefuji, Hiroki Aoyama, Masanori Nakayama, Saeko Kawabata, Yoji Kawano, Akihiro Iwamatsu, Mutsuki Amano, Kozo Kaibuchi

Genes to cells : devoted to molecular & cellular mechanisms 10 ( 10 ) 973 - 87 2005.10

Language：English Publishing type：Research paper (scientific journal)

After binding of epidermal growth factor (EGF), the EGF receptor is activated, internalized by endocytosis, and subsequently degraded in the lysosomal pathway. Endocytotic trafficking of the activated EGF receptor is essential for controlling EGF signaling. Upon ligand-induced activation of EGF receptors, Cbl (ubiquitin ligase) binds to the activated receptor and leads to translocation of the CIN85 (Cbl-interacting protein of 85 kDa)/endophilin complex in the vicinity of the activated EGF receptors. Endophilin is known as a key regulator of clathrin-mediated endocytosis, and the translocation of endophilin in the vicinity of active EGF receptor is thought to promote receptor internalization. The constitutively active mutant of small GTPase Rho inhibits EGF receptor endocytosis. In this study, we found that this inhibitory effect was canceled by the dominant negative form of Rho-associated kinase (Rho-kinase), which is an effector of Rho. To clarify the molecular mechanisms of endocytosis downstream of Rho/Rho-kinase signal, we searched for and identified endophilin A1 as a novel substrate of Rho-kinase. We identified the phosphorylation site of endophilin A1 at Thr-14 and made endophilin T14D (substitution of Thr-14 by Asp), which is expected to mimic the phosphorylation state of endophilin A1. Endophilin T14D inhibited EGF receptor internalization. Furthermore, phosphorylation of endophilin by Rho-kinase inhibited the binding to CIN85. Taken together, these results suggest that Rho-kinase phosphorylates endophilin downstream of Rho and regulates EGF receptor endocytosis through the inhibition of binding between endophilin and CIN85.

Rho-kinase and myosin II activities are required for cell type and environment specific migration. Reviewed International journal

Masanori Nakayama, Mutsuki Amano, Akira Katsumi, Takako Kaneko, Saeko Kawabata, Mikito Takefuji, Kozo Kaibuchi

Genes to cells : devoted to molecular & cellular mechanisms 10 ( 2 ) 107 - 17 2005.2

Language：English Publishing type：Research paper (scientific journal)

Cell migration is important in the development of atherosclerotic lesions. Macrophages and smooth muscle cells migrate into the subendothelial space of arteries, leading to plaque formation. Long-term inhibition of the activity of Rho-kinase induces a regression of atherosclerotic coronary lesions, probably by preventing migration of macrophages and smooth muscle cells. Previous reports concerning the effect of Rho-kinase inhibitors on cell migration are contradictory, however. We examined here the cell type specificity of Rho-kinase inhibitors and found that migration of endothelial cells, macrophages, and smooth muscle cells was inhibited by treatment with Rho-kinase inhibitors in a dose-dependent fashion in a three-dimensional migration assay, whereas that of fibroblasts and epithelial cells was not inhibited. Myosin II inhibitor prevented cell migration in a manner similar to Rho-kinase inhibitors. In contrast, in a two-dimensional migration assay, cell migration was not inhibited by Rho-kinase or myosin II inhibitors for any of the cell types examined. Taken together, these results indicate that Rho-kinase inhibitors suppress migration of specific cell types under specific conditions through the regulation of myosin II activity. Our findings suggest that Rho-kinase is the therapeutic target of atherosclerosis accompanied with invasion by leukocytes and smooth muscle cells.

[Involvement of small GTPase Rho and Rho-kinase in the pathogenesis of hypertension and hypertensive target organ damage]. Reviewed

Masanori Nakayama, Mutsuki Amano, Saeko Kawabata, Kozo Kaibuchi, Mikihito Takefuji

Nihon rinsho. Japanese journal of clinical medicine 62 Suppl 3 202 - 6 2004.3

Identification of Tau and MAP2 as novel substrates of Rho-kinase and myosin phosphatase. Reviewed International journal

Mutsuki Amano, Takako Kaneko, Akio Maeda, Masanori Nakayama, Masaaki Ito, Takashi Yamauchi, Hideyuki Goto, Yuko Fukata, Noriko Oshiro, Azusa Shinohara, Akihiro Iwamatsu, Kozo Kaibuchi

Journal of neurochemistry 87 ( 3 ) 780 - 90 2003.11

Language：English Publishing type：Research paper (scientific journal)

Rho-kinase and myosin phosphatase are implicated in the phosphorylation-state of myosin light chain downstream of Rho, which is thought to induce smooth muscle contraction and stress fibre formation in non-muscle cells. Here, we found that microtubule-associated proteins, Tau and MAP2, interacted with the myosin-binding subunit (MBS) of myosin phosphatase, and were the possible substrates of both Rho-kinase and myosin phosphatase. We determined the phosphorylation sites of Tau (Thr245, Thr377, Ser409) and MAP2 (Ser1796) by Rho-kinase. We also found that Rho-kinase phosphorylated Tau at Ser262 to some extent. Phosphorylation by Rho-kinase decreased the activity of Tau to promote microtubule assembly in vitro. Substitutions of Ala for Ser/Thr at the phosphorylation sites of Tau (Tau-AAA) did not affect the activity to promote microtubule assembly, while substitutions of Asp for Ser/Thr (Tau-DDD), which are expected to mimic the phosphorylation-state of Tau, slightly reduced the activity. When Tau, or mutated forms of Tau, were expressed in PC12 cells, followed by treatment with cytochalasin D, they promoted extension of the cell process in a cytochalasin-dependent manner. However, Tau-DDD showed the weaker activity in this capacity than wild-type Tau or Tau-AAA. These results suggest that the phosphorylation-state of these residues of Tau affects its activity both in vitro and in vivo. Thus, it is likely that the Rho-kinase/MBS pathway regulates not only the actin-myosin system but also microtubule dynamics.

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