Directory of Researchers

UEYAMA Takehiko
Biosignal Research Center
Professor
Medicine
Last Updated :2021/11/20

Researcher Profile and Settings

Affiliation

  • <Faculty / Graduate School / Others>

    Biosignal Research Center
  • <Related Faculty / Graduate School / Others>

    Graduate School of Medicine / Faculty of Biomedical Sciences, Graduate School of Medicine / Faculty of Medical Sciences

Research at Kobe

Research Activities

Research Interests

  • 1103
  • 6905
  • 6904
  • 5805

Research Areas

  • Life sciences / Medical biochemistry
  • Life sciences / Neuroscience - general
  • Life sciences / Pharmacology

Committee Memberships

  • 日本薬理学会, 評議員

Awards

  • Aug. 2005 広島大学医学部医学科広仁会, 広島大学医学部医学科広仁会 「平成17年度 基礎医学研究賞」, 新規NADPH oxidase (Nox4) の活性化機構と神経膠腫浸潤における役割の解明

    UEYAMA TAKEHIKO

    Others

Published Papers

  • Yuzuru Ninoyu, Hirofumi Sakaguchi, Chen Lin, Toshiaki Suzuki, Shigeru Hirano, Yasuo Hisa, Naoaki Saito, Takehiko Ueyama

    Dia1, which belongs to the diaphanous-related formin family, influences a variety of cellular processes through straight actin elongation activity. Recently, novel DIA1 mutants such as p.R1213X (p.R1204X) and p.A265S, have been reported to cause an autosomal dominant sensorineural hearing loss (DFNA1). Additionally, active DIA1 mutants induce progressive hearing loss in a gain-of-function manner. However, the subcellular localization and pathological function of DIA1(R1213X/R1204X) remains unknown. In the present study, we demonstrated the localization of endogenous Dia1 and the constitutively active DIA1 mutant in the cochlea, using transgenic mice expressing FLAG-tagged DIA1(R1204X) (DIA1-TG). Endogenous Dia1 and the DIA1 mutant were regionally expressed at the organ of Corti and the spiral ganglion from early life; alongside cochlear maturation, they became localized at the apical junctional complexes (AJCs) between hair cells (HCs) and supporting cells (SCs). To investigate HC vulnerability in the DIA1-TG mice, we exposed 4-week-old mice to moderate noise, which induced temporary threshold shifts with cochlear synaptopathy and ultrastructural changes in stereocilia 4 weeks post noise exposure. Furthermore, we established a knock-in (KI) mouse line expressing AcGFP-tagged DIA1(R1213X) (DIA1-KI) and confirmed mutant localization at AJCs and the tips of stereocilia in HCs. In MDCKAcGFP-DIA1(R1213X) cells with stable expression of AcGFP-DIA1(R1213X), AcGFP-DIA1(R1213X) revealed marked localization at microvilli on the apical surface of cells and decreased localization at cell-cell junctions. The DIA1-TG mice demonstrated hazy and ruffled circumferential actin belts at AJCs and abnormal stereocilia accompanied with HC loss at 5 months of age. In conclusion, Dia1 plays a pivotal role in the development and maintenance of AJCs and stereocilia, ensuring cochlear and HC integrity. Subclinical/latent vulnerability of HCs may be the cause of progressive hearing loss in DFNA1 patients, thus suggesting new therapeutic targets for preventing HC degeneration and progressive hearing loss associated with DFNA1.

    Corresponding, 16 Jul. 2020, Cell death & disease, 11 (7), 536 - 536, English, International magazine

    [Refereed]

    Scientific journal

  • Shigefumi Morioka, Hirofumi Sakaguchi, Hiroaki Mohri, Mariko Taniguchi-Ikeda, Motoi Kanagawa, Toshiaki Suzuki, Yuko Miyagoe-Suzuki, Tatsushi Toda, Naoaki Saito, Takehiko Ueyama

    Corresponding, Public Library of Science (PLoS), 26 May 2020, PLOS Genetics, 16 (5), e1008826 - e1008826, English

    [Refereed]

    Scientific journal

  • UEYAMA TAKEHIKO, Megumi Sakuma, Mio Nakatsuji, Tatsuya Uebi, Takeshi Hamada, Atsu Aiba, SAITO NAOAKI

    Jan. 2020, J Investigative Dermatology, 140, 75 - 84, English

    [Refereed]

    Scientific journal

  • Differential disruption of autoinhibition and defect in assembly of cytoskeleton during cell division decide the fates of human DIAPH1-related cytoskeletopathy

    Bong Jik Kim, UEYAMA TAKEHIKO, Takushi Miyoshi, Shin-ichiro Kitajiri, Byung Yoon Choi

    Dec. 2019, J Medical Genetics, 56 (12), 818 - 827, English

    [Refereed]

    Scientific journal

  • Shirafuji T, Shimazaki H, Miyagi T, Ueyama T, Adachi N, Tanaka S, Hide I, Saito N, Sakai N.

    Spinocerebellar ataxia type 14 (SCA14) is an autosomal dominant neurodegenerative disorder characterized by cerebellar ataxia with myoclonus, dystonia, spasticity, and rigidity. Although missense mutations and a deletion mutation have been found in the protein kinase C gamma (PRKCG) gene encoding protein kinase C γ (PKCγ) in SCA14 families, a nonsense mutation has not been reported. The patho-mechanisms underlying SCA14 remain poorly understood. However, gain-of-function mechanisms and loss-of-function mechanisms, but not dominant negative mechanisms, were reported the patho-mechanism of SCA14. We identified the c.226C>T mutation of PRKCG, which caused the p.R76X in PKCγ by whole-exome sequencing in patients presenting cerebellar atrophy with cognitive and hearing impairment. To investigate the patho-mechanism of our case, we studied aggregation formation, cell death, and PKC inhibitory effect by confocal microscopy, western blotting with cleaved caspase 3, and pSer PKC motif antibodies, respectively. PKCγ(R76X)-GFP have aggregations the same as wild-type (WT) PKCγ-GFP. The PKCγ(R76X)-GFP inhibited PKC phosphorylation activity more than GFP alone. It also induced more apoptosis in COS7 and SH-SY5Y cells compared to WT-PKCγ-GFP and GFP. We first reported SCA14 patients with p.R76X in PKCγ who have cerebellar atrophy with cognitive and hearing impairment. Our results suggest that a dominant negative mechanism due to truncated peptides produced by p.R76X may be at least partially responsible for the cerebellar atrophy.

    Jul. 2019, Molecular and Cellular Neuroscience, 98, 46 - 53, English, International magazine

    [Refereed]

    Scientific journal

  • Nakazono A, Adachi Naoko, Takahashi H, Seki T, Hamada D, Ueyama Takehiko, Sakai N, Saito Naoaki

    Amyloid and amyloid-like protein aggregations are hallmarks of multiple, varied neurodegenerative disorders, including Alzheimer’s and Parkinson’s diseases. We previously reported that spinocerebellar ataxia type 14 (SCA14), a dominant-inherited neurodegenerative disease that affects cerebellar Purkinje cells, is characterized by the intracellular formation of neurotoxic amyloid-like aggregates of genetic variants of protein kinase Cɣ (PKCɣ). A number of protein chaperones, including heat shock protein 70 (Hsp70), promote the degradation and/or refolding of misfolded proteins and thereby prevent their aggregation. Here, we report that, in various SCA14-associated, aggregating PKCɣ variants, endogenous Hsp70 is incorporated into aggregates and that expression of these PKCɣ mutants up-regulates Hsp70 expression. We observed that PKCɣ binds Hsp70 and that this interaction is enhanced in the SCA14-associated variants, mediated by the kinase domain that is involved in amyloid-like fibril formation as well as the C2 domain of PKCɣ. Pharmacological up-regulation of Hsp70 by the Hsp90 inhibitors celastrol and herbimycin A attenuated the aggregation of mutant PKCɣ in primary cultured Purkinje cells. Up-regulation of Hsp70 diminished net PKCɣ aggregation by preventing aggregate formation, resulting in decreased levels of apoptotic cell death among primary cultured Purkinje cells expressing the PKCɣ variant. Of note, herbimycin A also ameliorated abnormal dendritic development. Extending our in vitro observations, administration of celastrol to mice up-regulated cerebellar Hsp70. Our findings identify heat shock proteins as important endogenous regulators of pathophysiological PKCɣ aggregation and point to Hsp90 inhibition as a potential therapeutic strategy in the treatment of SCA14.

    Sep. 2018, J Biol Chem, 293 (38), 14758 - 14774, English

    [Refereed]

    Scientific journal

  • MORIOKA SHIGEFUMI, SAKAGUCHI HIROFUMI, Yamaguchi T, Ninoyu Y, Mohri H, Nakamura T, Hisa Y, Ogita K, SAITO NAOAKI, UEYAMA TAKEHIKO

    Previous studies have convincingly argued that reactive oxygen species (ROS) contribute to the development of several major types of sensorineural hearing loss, such as noise-induced hearing loss (NIHL), drug-induced hearing loss, and age-related hearing loss. However, the underlying molecular mechanisms induced by ROS in these pathologies remain unclear. To resolve this issue, we established an in vivo model of ROS overproduction by generating a transgenic (TG) mouse line expressing the human NADPH oxidase 4 (NOX4, NOX4-TG mice), which is a constitutively active ROS-producing enzyme that does not require stimulation or an activator. Overproduction of ROS was detected at the cochlea of the inner ear in NOX4-TG mice, but they showed normal hearing function under baseline conditions. However, they demonstrated hearing function vulnerability, especially at high-frequency sounds, upon exposure to intense noise, which was accompanied by loss of cochlear outer hair cells (OHCs). The vulnerability to loss of hearing function and OHCs was rescued by treatment with the antioxidant Tempol. Additionally, we found increased protein levels of the heat-shock protein 47 (HSP47) in models using HEK293 cells, including H2 O2 treatment and cells with stable and transient expression of NOX4. Furthermore, the up-regulated levels of Hsp47 were observed in both the cochlea and heart of NOX4-TG mice. Thus, antioxidant therapy is a promising approach for the treatment of NIHL. Hsp47 may be an endogenous antioxidant factor, compensating for the chronic ROS overexposure in vivo, and counteracting ROS-related hearing loss.

    Corresponding, Aug. 2018, J Neurochemistry, 146 (4), 459 - 473, English, International magazine

    [Refereed]

    International conference proceedings

  • Isao Sakamoto, Takehiko Ueyama, Masakazu Hayashibe, Takashi Nakamura, Hiroaki Mohri, Hiroshi Kiyonari, Michiko Shigyo, Chihiro Tohda, Naoaki Saito

    Bergmann glia (BG) are important in the inward type of radial migration of cerebellar granule neurons (CGNs). However, details regarding the functions of Cdc42 and Rac in BG for radial migration of CGN are unknown. To examine the roles of Cdc42 and Rac in BG during cerebellar corticogenesis, mice with a single deletion of Cdc42 or Rac1 and those with double deletions of Cdc42 and Rac1 under control of the glial fibrillary acidic protein (GFAP) promoter: GFAP-Cre Cdc42flox/flox (Cdc42-KO), GFAP–Cre Rac1flox/flox (Rac1-KO), and GFAP-Cre Cdc42 flox/flox Rac1flox/flox (Cdc42/Rac1-DKO) mice, were generated. Both Cdc42-KO and Rac1-KO mice, but more obviously Cdc42-KO mice, had disturbed alignment of BG in the Purkinje cell layer (PCL). We found that Cdc42-KO, but not Rac1-KO, induced impaired radial migration of CGNs in the late phase of radial migration, leading to retention of CGNs in the lower half of the molecular layer (ML). Cdc42-KO, but not Rac1-KO, mice also showed aberrantly aligned Purkinje cells (PCs). These phenotypes were exacerbated in Cdc42/Rac1-DKO mice. Alignment of BG radial fibers in the ML and BG endfeet at the pial surface of the cerebellum evaluated by GFAP staining was disturbed and weak in Cdc42/Rac1-DKO mice, respectively. Our data indicate that Cdc42 and Rac, but predominantly Cdc42, in BG play important roles during the late phase of radial migration of CGNs. We also report here that Cdc42 is involved in gliophilic migration of CGNs, in contrast to Rac, which is more closely connected to regulating neurophilic migration.

    Academic Press Inc., 01 Apr. 2018, Experimental Neurology, 302, 57 - 67, English

    [Refereed]

    Scientific journal

  • Toshihiko Shirafuji, Takehiko Ueyama, Naoko Adachi, Ken-Ichi Yoshino, Yusuke Sotomaru, Junsuke Uwada, Azumi Kaneoka, Taro Ueda, Shigeru Tanaka, Izumi Hide, Naoaki Saito, Norio Sakai

    Protein kinase Cγ (PKCγ) knock-out (KO) animals exhibit symptoms of Parkinson’s disease (PD), including dopaminergic neuronal loss in the substantia nigra. However, the PKCγ substrates responsible for the survival of dopaminergic neurons in vivo have not yet been elucidated. Previously, we found 10 potent substrates in the striatum of PKCγ-KO mice. Here, we focused on cysteine string protein α (CSPα), a protein from the heat shock protein (HSP) 40 cochaperone families localized on synaptic vesicles. We found that in cultured cells, PKCγ phosphorylates CSPα at serine (Ser) 10 and Ser34. Additionally, apoptosis was found to have been enhanced by the overexpression of a phosphorylation-null mutant of CSPα, CSPα(S10A/S34A). Compared with wild-type (WT) CSPα, the CSPα(S10A/S34A) mutant had a weaker interaction with HSP70. However, in sharp contrast, a phosphomimetic CSPα(S10D/S34D) mutant, compared with WT CSPα, had a stronger interaction with HSP70. In addition, total levels of synaptosomal-associated protein (SNAP) 25, a main downstream target of the HSC70/HSP70 chaperone complex, were found to have decreased by the CSPα(S10A/S34A) mutant through increased ubiquitination of SNAP25 in PC12 cells. In the striatum of 2-year-old male PKCγ-KO mice, decreased phosphorylation levels of CSPα and decreased SNAP25 protein levels were observed. These findings indicate the phosphorylation of CSPα by PKCγ may protect the presynaptic terminal from neurodegeneration. The PKCγ–CSPα–HSC70/HSP70–SNAP25 axis, because of its role in protecting the presynaptic terminal, may provide a new therapeutic target for the treatment of PD.

    Society for Neuroscience, 10 Jan. 2018, Journal of Neuroscience, 38 (2), 278 - 290, English

    [Refereed]

    Scientific journal

  • Cheryl M. Hanes, Anna E. D'Amico, Takehiko Ueyama, Alexander C. Wong, Xuexin Zhang, W. Frederick Hynes, Margarida M. Barroso, Nathaniel C. Cady, Mohamed Trebak, Naoaki Saito, Michelle R. Lennartz

    Protein kinase C-epsilon (PKC-epsilon) at phagocytic cups mediates the membrane fusion necessary for efficient IgG-mediated phagocytosis. The C1B and pseudosubstrate (epsilon PS) domains are necessary and sufficient for this concentration. C1B binds diacylglycerol; the docking partner for epsilon PS is unknown. Liposome assays revealed that the epsilon PS binds phosphatidylinositol 4-phosphate (PI4P) and PI(3,5) P-2. Wortmannin, but not LY294002, inhibits PKC-epsilon concentration at cups and significantly reduces the rate of phagocytosis. As Wortmannin inhibits PI4 kinase, we hypothesized that PI4P mediates the PKC-epsilon concentration at cups and the rate of phagocytosis. PKC-epsilon colocalizes with the trans-Golgi network (TGN) PI4P reporter, P4M, suggesting it is tethered at the TGN. Real-time imaging of GFP-PKC-epsilon -expressing macrophages revealed a loss of Golgi-associated PKC-epsilon during phagocytosis, consistent with a Golgi-to-phagosome translocation. Treatment with PIK93, a PI4 kinase inhibitor, reduces PKC-epsilon at both the TGN and the cup, decreases phagocytosis, and prevents the increase in capacitance that accompanies membrane fusion. Finally, expression of the Golgi-directed PI4P phosphatase, hSac1-K2A, recapitulates the PIK93 phenotype, confirming that Golgiassociated PI4P is critical for efficient phagocytosis. Together these data are consistent with a model in which PKC-epsilon is tethered to the TGN via an epsilon PS-PI4P interaction. The TGN-associated pool of PKC-epsilon concentrates at the phagocytic cup where it mediates the membrane fusion necessary for phagocytosis. The novelty of these data lies in the demonstration that epsilon PS binds PI4P and PI(3,5) P-2 and that PI4P is necessary for PKC-epsilon localization at the TGN, its translocation to the phagocytic cup, and the membrane fusion required for efficient Fc [gamma] receptor-mediated phagocytosis.

    AMER ASSOC IMMUNOLOGISTS, Jul. 2017, JOURNAL OF IMMUNOLOGY, 199 (1), 271 - 277, English

    [Refereed]

    Scientific journal

  • Takashi Nakamura, Takehiko Ueyama, Yuzuru Ninoyu, Hirofumi Sakaguchi, Narantsog Choijookhuu, Yoshitaka Hishikawa, Hiroshi Kiyonari, Masaaki Kohta, Mizuho Sakahara, Ivan de Curtis, Eiji Kohmura, Yasuo Hisa, Atsu Aiba, Naoaki Saito

    Rac signaling impacts a relatively large number of downstream targets; however, few studies have established an association between Rac pathways and pathological conditions. In the present study, we generated mice with double knockout of Rac1 and Rac3 (Atoh1-Cre;Rac1flox/flox;Rac3-/- ) in cerebellar granule neurons (CGNs). We observed impaired tangential migration at E16.5, as well as numerous apoptotic CGNs at the deepest layer of the external granule layer (EGL) in the medial cerebellum of Atoh1-Cre;Rac1flox/flox;Rac3-/- mice at P8. Atoh1-Cre;Rac1flox/flox;Rac3-/- CGNs differentiated normally until expression of p27kip1 and NeuN in the deep EGL at P5. Primary CGNs and cerebellar microexplants from Atoh1-Cre;Rac1flox/flox;Rac3-/- mice exhibited impaired neuritogenesis, which was more apparent in Map2-positive dendrites. Such findings suggest that impaired tangential migration and final differentiation of CGNs have resulted in decreased cerebellum size and agenesis of the medial internal granule layer, respectively. Furthermore, Rac depleted/deleted cells exhibited decreased levels of Mid1 and impaired mTORC1 signaling. Mid1 depletion in CGNs produced mild impairments in neuritogenesis and reductions in mTORC1 signaling. Thus, a novel Rac-signaling pathway (Rac1-Mid1-mTORC1) may be involved in medial cerebellar development.

    15 May 2017, Development (Cambridge, England), 144 (10), 1863 - 1875, English, International magazine

    [Refereed]

    Scientific journal

  • Taiji Ishii, Takehiko Ueyama, Michiko Shigyo, Masaaki Kohta, Takeshi Kondoh, Tomoharu Kuboyama, Tatsuya Uebi, Takeshi Hamada, David H Gutmann, Atsu Aiba, Eiji Kohmura, Chihiro Tohda, Naoaki Saito

    Astrogliosis (i.e. glial scar), which is comprised primarily of proliferated astrocytes at the lesion site and migrated astrocytes from neighboring regions, is one of the key reactions in determining outcomes after CNS injury. In an effort to identify potential molecules/pathways that regulate astrogliosis, we sought to determine whether Rac/Rac-mediated signaling in astrocytes represents a novel candidate for therapeutic intervention following CNS injury. For these studies, we generated mice with Rac1 deletion under the control of the GFAP (glial fibrillary acidic protein) promoter (GFAP-Cre;Rac1flox/flox). GFAP-Cre;Rac1flox/flox (Rac1-KO) mice exhibited better recovery after spinal cord injury and exhibited reduced astrogliosis at the lesion site relative to control. Reduced astrogliosis was also observed in Rac1-KO mice following microbeam irradiation-induced injury. Moreover, knockdown (KD) or KO of Rac1 in astrocytes (LN229 cells, primary astrocytes, or primary astrocytes from Rac1-KO mice) led to delayed cell cycle progression and reduced cell migration. Rac1-KD or Rac1-KO astrocytes additionally had decreased levels of GSPT1 (G1 to S phase transition 1) expression and reduced responses of IL-1β and GSPT1 to LPS treatment, indicating that IL-1β and GSPT1 are downstream molecules of Rac1 associated with inflammatory condition. Furthermore, GSPT1-KD astrocytes had cell cycle delay, with no effect on cell migration. The cell cycle delay induced by Rac1-KD was rescued by overexpression of GSPT1. Based on these results, we propose that Rac1-GSPT1 represents a novel signaling axis in astrocytes that accelerates proliferation in response to inflammation, which is one important factor in the development of astrogliosis/glial scar following CNS injury.

    27 Jan. 2017, The Journal of biological chemistry, 292 (4), 1240 - 1250, English, International magazine

    [Refereed]

    Scientific journal

  • Hirofumi Sakaguchi, Takashi Nakamura, Takehiko Ueyama

    The inner ear consists of the cochlea and vestibule, both containing sensory hair cells, which possess strictly regulated actin organization to conduct sensitive mechanoelectrical transduction. Rho-GTPase is a family of small GTPases, known to play key roles in actin regulation. CDC 42 and Rac (Rac 1, Rac 2, Rac 3) are major members of Rho-GTPase family and we have previously confirmed the expression of CDC 42, Rac 1 and Rac 3 in cochlear sensory epithelia. Recently we studied the function of CDC 42 and Rac in inner ears and the cerebellum using a gene-targeting strategy. We developed a CDC 42-conditional knock-out mouse (CDC 42-CKO mouse) using the Cre-loxP system under an Atoh 1 promotor to establish a model of hair cell-specific deletion of CDC 42. The CDC 42-CKO mouse showed progressive hearing loss associated with hair bundle degeneration, structural abnormality of the apical cell junction and hair cell loss. On the other hand, the Rac 1-CKO mouse, Rac 3-KO mouse and their crossbred Rac 1/Rac 3 double knock-out (Rac 1/3-DKO) mouse did not show this hearing phenotype. These data indicated that CDC 42 is essential for maintaining hearing function by regulation of actin dynamics in the hair cells, whereas Rac molecules are not necessary for hair cell function. Both the CDC 42-CKO mouse and Rac 1/3-DKO did not show the vestibular phenotype, suggesting the existence of other Rho-GTPases compensating for the function of CDC 42 in the vestibular organ. Moreover, the Rac 1/3-DKO mouse showed an ataxic gait and cerebellar hypoplasia, although these phenotypes are much more subtle in the Rac 1-KO mouse and not observed in the Rac 3-KO mouse. We found hypoplasia of the inner granular layer and dysfunction of neurite growth in the Rac 1/3-DKO mouse, suggesting a compensatory function of Rac 1 and Rac 3 in the neurite extension and cellular migration of cerebellar granular cells. Further studies are needed to explore the function and compensatory mechanism of the Rho-GTPase family in the inner ears and cerebellum to elucidate fully the molecular regulation of the hearing and balance system.

    Japan Society for Equilibrium Research, 2017, Equilibrium Research, 76 (6), 720 - 726, Japanese

    [Refereed]

    Scientific journal

  • Toshihiko Shirafuji, Takehiko Ueyama, Shigeru Tanaka, Izumi Hide, Naoaki Saito, Norio Sakai

    There are many commercial antibodies with little information provided by their suppliers as to their reliability. Accordingly, commercial antibodies require proper validation before being used in scientific research. In this study, we validated several commercial antibodies, including anti-CSP alpha, SNAP25, tyrosine hydroxylase, ubiquitin, cleaved caspase 3, and pSer PKC motif. Anti-CSP alpha, SNAP25, and tyrosine hydroxylase antibodies could detect their endogenous target proteins with some degree of cross-reactivity. Furthermore, clear SNAP25 staining was observed with SNAP25 antibody. Antibodies directed against ubiquitin, cleaved caspase 3, and pSer PKC motif could detect poly-ubiquitination, apoptosis, and phosphorylation, respectively.

    JAPAN SOC HISTOCHEMISTRY & CYTOCHEMISTRY, 2017, ACTA HISTOCHEMICA ET CYTOCHEMICA, 50 (6), 177 - 180, English

    [Refereed]

    Scientific journal

  • Takehiko Ueyama, Yuzuru Ninoyu, Shin-ya Nishio, Takushi Miyoshi, Hiroko Torii, Koji Nishimura, Kazuma Sugahara, Hideaki Sakata, Dean Thumkeo, Hirofumi Sakaguchi, Naoki Watanabe, Shin-ichi Usami, Naoaki Saito, Shin-ichiro Kitajiri

    DIAPH1 encodes human DIA1, a formin protein that elongates unbranched actin. The c.3634+1G>T DIAPH1 mutation causes autosomal dominant nonsyndromic sensorineural hearing loss, DFNA1, characterized by progressive deafness starting in childhood. The mutation occurs near the C-terminus of the diaphanous autoregulatory domain (DAD) of DIA1, which interacts with its N-terminal diaphanous inhibitory domain (DID), and may engender constitutive activation of DIA1. However, the underlying pathogenesis that causes DFNA1 is unclear. We describe a novel patient-derived DIAPH1 mutation (c.3610C>T) in two unrelated families, which results in early termination prior to a basic amino acid motif (RRKR1204-1207) at the DAD C-terminus. The mutant DIA1(R1204X) disrupted the autoinhibitory DID-DAD interaction and was constitutively active. This unscheduled activity caused increased rates of directional actin polymerization movement and induced formation of elongated microvilli. Mice expressing FLAG-tagged DIA1(R1204X) experienced progressive deafness and hair cell loss at the basal turn and had various morphological abnormalities in stereocilia (short, fused, elongated, sparse). Thus, the basic region of the DAD mediates DIA1 autoinhibition; disruption of the DID-DAD interaction and consequent activation of DIA1(R1204X) causes DFNA1.

    WILEY-BLACKWELL, Nov. 2016, EMBO MOLECULAR MEDICINE, 8 (11), 1310 - 1324, English

    [Refereed]

    Scientific journal

  • Takehiko Ueyama, Megumi Sakuma, Yuzuru Ninoyu, Takeshi Hamada, Corinne Dupuy, Miklós Geiszt, Thomas L. Leto, Naoaki Saito

    NADPH oxidase (Nox) family proteins produce superoxide (O2 .-) directly by transferring an electron to molecular oxygen. Dual oxidases (Duoxes) also produce an O2 .- intermediate, although the final species secreted by mature Duoxes is H2O2, suggesting that intramolecular O2 .- dismutation or other mechanisms contribute to H2O2 release. We explored the structural determinants affecting reactive oxygen species formation by Duox enzymes. Duox2 showed O2 .- leakage when mismatched with Duox activator 1 (DuoxA1). Duox2 released O2 .- even in correctly matched combinations, including Duox2 + DuoxA2 and Duox2 + N-terminally tagged DuoxA2 regardless of the type or number of tags. Conversely, Duox1 did not release O2 .- in any combination. Chimeric Duox2 possessing the A-loop of Duox1 showed no O2 .- leakage chimeric Duox1 possessing the A-loop of Duox2 released O2 .-. Moreover, Duox2 proteins possessing the A-loops of Nox1 or Nox5 co-expressed with DuoxA2 showed enhanced O2 .- release, and Duox1 proteins possessing the A-loops of Nox1 or Nox5 co-expressed with DuoxA1 acquired O2 .- leakage. Although we identified Duox1 A-loop residues (His1071, His1072, and Gly1074) important for reducing O2 .- release, mutations of these residues to those of Duox2 failed to convert Duox1 to an O2 .- releasing enzyme. Using immunoprecipitation and endoglycosidase H sensitivity assays, we found that the A-loop of Duoxes binds to DuoxA N termini, creating more stable, mature Duox-DuoxA complexes. In conclusion, the A-loops of both Duoxes support H2O2 production through interaction with corresponding activators, but complex formation between the Duox1 A-loop and DuoxA1 results in tighter control of H2O2 release by the enzyme complex.

    American Society for Biochemistry and Molecular Biology Inc., 06 Mar. 2015, Journal of Biological Chemistry, 290 (10), 6495 - 6506, English

    [Refereed]

    Scientific journal

  • Hideyuki Takahashi, Naoko Adachi, Toshihiko Shirafuji, Sally Danno, Takehiko Ueyama, Michele Vendruscolo, Anton N. Shuvaev, Takuya Sugimoto, Takahiro Seki, Daizo Hamada, Kazuhiro Irie, Hirokazu Hirai, Norio Sakai, Naoaki Saito

    Amyloid assemblies are associated with a wide range of human disorders, including Alzheimer's and Parkinson's diseases. Here, we identify protein kinase C (PKC) gamma, a serine/threonine kinase mutated in the neurodegenerative disease spinocerebellar ataxia type 14 (SCA14), as a novel amyloidogenic protein with no previously characterized amyloid-prone domains. We found that overexpression of PKC gamma in cultured cells, as well as in vitro incubation of PKC gamma without heat or chemical denaturants, causes amyloid-like fibril formation of this protein. We also observed that SCA14-associated mutations in PKC gamma accelerate the amyloid-like fibril formation both in cultured cells and in vitro. We show that the C1A and kinase domains of PKC gamma are involved in its soluble dimer and aggregate formation and that SCA14-associated mutations in the Cl domain cause its misfolding and aggregation. Furthermore, long-term time-lapse imaging indicates that aggregates of mutant PKC gamma are highly toxic to neuronal cells. Based on these findings, we propose that PKC gamma could form amyloid-like fibrils in physiological and/or pathophysiological conditions such as SCA14. More generally, our results provide novel insights into the mechanism of amyloid-like fibril formation by multi-domain proteins.

    OXFORD UNIV PRESS, Jan. 2015, HUMAN MOLECULAR GENETICS, 24 (2), 525 - 539, English

    [Refereed]

    Scientific journal

  • RANTES/CCL5 mediated-biological effects depend on the syndecan-4/PKCα signaling pathway

    Maillard L, Saito Naoaki, Hlawaty H, Friand V, Suffee N, Chmilewsky F, Haddad O, Laguillier C, Guyot E, Ueyama Takehiko, Oudar O, Sutton A, Charnaux N

    Sep. 2014, Biol Open, 3 (10), 995 - 1004, English

    [Refereed]

    Scientific journal

  • Toshihiko Shirafuji, Takehiko Ueyama, Ken-ichi Yoshino, Hideyuki Takahashi, Naoko Adachi, Yukio Ago, Ken Koda, Tetsuaki Nashida, Naoki Hiramatsu, Toshio Matsuda, Tatsushi Toda, Norio Sakai, Naoaki Saito

    Protein kinase C (PKC) has been implicated in the control of neurotransmitter release. The AS/AGU rat, which has a nonsense mutation in PKC gamma, shows symptoms of parkinsonian syndrome, including dopamine release impairments in the striatum. Here, we found that the AS/AGU rat is PKC gamma-knock-out (KO) and that PKC gamma-KO mice showed parkinsonian syndrome. However, the PKC gamma substrates responsible for the regulated exocytosis of dopamine in vivo have not yet been elucidated. To identify the PKC gamma substrates involved in dopamine release, we used PKC gamma-KO mice and a phosphoproteome analysis. We found 10 candidate phosphoproteins that had decreased phosphorylation levels in the striatum of PKC gamma-KO mice. We focused on Pak-interacting exchange factor-beta(beta PIX), a Cdc42/Rac1 guanine nucleotide exchange factor, and found that PKC gamma directly phosphorylates beta PIX at Ser583 and indirectly at Ser340 in cells. Furthermore, we found that PKC phosphorylated beta PIX in vivo. Classical PKC inhibitors and beta PIX knock-down (KD) significantly suppressed Ca2+ evoked dopamine release in PC12 cells. Wild-type beta PIX, and not the beta PIX mutants Ser340 Ala or Ser583 Ala, fully rescued the decreased dopamine release by beta PIX KD. Double KD of Cdc42 and Rac1 decreased dopamine release from PC12 cells. These findings indicate that the phosphorylation of beta PIX at Ser340 and Ser583 has pivotal roles in Ca2+-evoked dopamine release in the striatum. Therefore, we propose that PKC gamma positively modulates dopamine release through beta 2PIX phosphorylation. The PKC gamma-beta PIX-Cdc42/Rac1 phosphorylation axis may provide a new therapeutic target for the treatment of parkinsonian syndrome.

    SOC NEUROSCIENCE, Jul. 2014, JOURNAL OF NEUROSCIENCE, 34 (28), 9268 - 9280, English

    [Refereed]

    Scientific journal

  • Takehiko Ueyama, Hirofumi Sakaguchi, Takashi Nakamura, Akihiro Goto, Shigefumi Morioka, Aya Shimizu, Kazuki Nakao, Yoshitaka Hishikawa, Yuzuru Ninoyu, Hidetoshi Kassai, Shiro Suetsugu, Takehiko Koji, Bernd Fritzsch, Shigenobu Yonemura, Yasuo Hisa, Michiyuki Matsuda, Atsu Aiba, Naoaki Saito

    Cdc42 is a key regulator of dynamic actin organization. However, little is known about how Cdc42-dependent actin regulation influences steady-state actin structures in differentiated epithelia. We employed inner ear hair-cell-specific conditional knockout to analyze the role of Cdc42 in hair cells possessing highly elaborate stable actin protrusions (stereocilia). Hair cells of Atoh1-Cre; Cdc42(flox/flox) mice developed normally but progressively degenerated after maturation, resulting in progressive hearing loss particularly at high frequencies. Cochlear hair cell degeneration was more robust in inner hair cells than in outer hair cells, and began as stereocilia fusion and depletion, accompanied by a thinning and waving circumferential actin belt at apical junctional complexes (AJCs). Adenovirus-encoded GFP-Cdc42 expression in hair cells and fluorescence resonance energy transfer (FRET) imaging of hair cells from transgenic mice expressing a Cdc42-FRET biosensor indicated Cdc42 presence and activation at stereociliary membranes and AJCs in cochlear hair cells. Cdc42-knockdown in MDCK cells produced phenotypes similar to those of Cdc42-deleted hair cells, including abnormal microvilli and disrupted AJCs, and downregulated actin turnover represented by enhanced levels of phosphorylated cofilin. Thus, Cdc42 influenced the maintenance of stable actin structures through elaborate tuning of actin turnover, and maintained function and viability of cochlear hair cells.

    COMPANY OF BIOLOGISTS LTD, May 2014, JOURNAL OF CELL SCIENCE, 127 (9), 2040 - 2052, English

    [Refereed]

    Scientific journal

  • Hyun Choi, Ju-yearl Park, Hyoung-June Kim, Minsoo Noh, Takehiko Ueyama, Yunsoo Bae, Tae Ryong Lee, Dong Wook Shin

    Background: Recent studies have demonstrated that the production of reactive oxygen species (ROS) itself plays an indispensable role in the process of differentiation in various tissues. However, it is unclear whether ROS have an effect on the differentiation of keratinocytes essential for the development of the epidermal permeability barrier. Objective: The aim of the study is to determine a major H2O2-generating source by ionomycin in normal human keratinocytes (NHKs), and elucidate the physiological role of H2O2 generated by identified dual oxidase 1 (DUOX1) on differentiation markers of NHKs. Methods: To detect H2O2 level generated by ionomycin in NHKs, luminal-HRP assays are performed. To examine the effects of DUOX1 on differentiation markers of NHKs, analysis of Q-RT-PCR, siRNA knockdown, and Western blot analysis were performed. Results: We found that levels of H2O2 generated by ionomycin, a Ca2+ signal inducer, showed Ca2+ dependence manner. In addition, DPI, an inhibitor of NOXes, significantly reversed the ionomycin-induced H2O2 level, and inhibited the mRNA expression levels of keratin 1, keratin 10, and filaggrin compared with other ROS generating system inhibitors. Interestingly, we demonstrated that extracellular Ca2+ markedly up-regulated mRNA expression levels of DUOX1 among NADPH oxidase (NOX) isoforms. Knockdown of DUOX1 by RNA interference (RNAi) in NHKs significantly antagonized an increase of ionomycin-induced H2O2 level, and specifically decreased the expressions of several keratinocyte differentiation markers such as keratin 1, transglutaminase 3, desmoglein 1, and aquaporin 9. In addition, we also found that formation of cornified envelope was significantly reduced in DUOX1-knockdown NHKs. Conclusion: These results suggest that DUOX1 is the major H2O2-producing source in NHKs stimulated with Ca2+, and plays a significant role in regulating the expression of specific markers necessary for the normal differentiation of keratinocytes. (c) 2013 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.

    ELSEVIER IRELAND LTD, Apr. 2014, JOURNAL OF DERMATOLOGICAL SCIENCE, 74 (1), 56 - 63, English

    [Refereed]

    Scientific journal

  • Megumi Sakuma, Yasuhito Shirai, Takehiko Ueyama, Naoaki Saito

    Diacylglycerol (DAG) is an important lipid that acts as a signaling messenger during mast cell degranulation after allergen cross-linking of immunoglobulin (Ig) E-bound REM receptors. In this study, we determined the role of diacylglycerol kinase (DGK), which negatively regulates DAG-dependent signaling by converting DAG to phosphatidic acid (PA), in the regulation of mast cell degranulation. Treating RBL (rat basophilic leukemia)-2H3 mast cells with a type I DGK inhibitor significantly reduced antigen-induced degranulation and PA production. Among type I DGK isoforms, we observed that DGK alpha and DGK gamma mRNAs were expressed in RBL-2H3 mast cells using reverse transcription polymerase chain reaction. DGK gamma knockdown, but not DGK alpha, by isoform-specific short hairpin RNAs reduced mast cell degranulation and Ca2+ influxes from the extracellular environment. These results suggest that DGK gamma regulates mast cell degranulation after REM cross-linking through mobilization of intracellular Ca2+ through Ca2+ influxes. (C) 2014 Elsevier Inc. All rights reserved.

    ACADEMIC PRESS INC ELSEVIER SCIENCE, Mar. 2014, BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 445 (2), 340 - 345, English

    [Refereed]

    Scientific journal

  • Takehiko Ueyama, Jeonghyun Son, Takeshi Kobayashi, Takeshi Hamada, Takashi Nakamura, Hirofumi Sakaguchi, Toshihiko Shirafuji, Naoaki Saito

    In its resting state, Rho GDP-dissociation inhibitor (RhoGDI) alpha forms a soluble cytoplasmic heterodimer with the GDP-bound form of Rac. Upon stimulation, the dissociation of RhoGDI alpha from the RhoGDI alpha-Rac complex is a mandatory step for Rac activation; however, this mechanism is poorly understood. In this study, we examined how the cytoplasm/membrane cycles of the RhoGDI-Rac complex are regulated, as well as where RhoGDI dissociates from the RhoGDI-Rac complex, during Fc gamma R-mediated phagocytosis. The negatively charged and flexible N terminus (25 residues) of RhoGDI alpha, particularly its second negative amino acid cluster possessing five negatively charged amino acids, was a pivotal regulator in the cytoplasm/membrane cycles of the RhoGDI-Rac complex. We also found that RhoGDI alpha translocated to the phagosomes as a RhoGDI alpha-Rac1 complex, and this translocation was mediated by an interaction between the polybasic motif in the C terminus of Rac1 and anionic phospholipids produced on phagosomes, such as phosphatidic acid, that is, by a phagosome-targeting mechanism of Rac1. Thus, we demonstrated that the targeting/accumulation of the RhoGDI alpha-Rac1 complex to phagosomes is regulated by a balance between three factors: 1) the negatively charged and flexible N-terminal of RhoGDI alpha, 2) the binding affinity of RhoGDI alpha for Rac1, and 3) anionic phospholipids produced on phagosomes. Moreover, we demonstrated that the mechanism of targeting/accumulation of the RhoGDI alpha-Rac1 complex is also applicable for the RhoGDI beta-Rac1 complex.

    AMER ASSOC IMMUNOLOGISTS, Sep. 2013, JOURNAL OF IMMUNOLOGY, 191 (5), 2560 - 2569, English

    [Refereed]

    Scientific journal

  • Daniel Schlam, Michal Bohdanowicz, Alexandros Chatilialoglu, Benjamin E. Steinberg, Takehiko Ueyama, Guangwei Du, Sergio Grinstein, Gregory D. Fairn

    Background: Cell population-based studies obscure potential phagosomal heterogeneity. Results:Weused a dynamic assay to monitor superoxide production in single phagosomes and uncovered variability inNADPH oxidase activity. Conclusion: The heterogeneity is attributable to variations in local DAG accumulation, which is controlled by DAG kinases. Significance: Heterogeneity in phagosome responsiveness could enable the survival of a fraction of invading microorganisms. © 2013 by The American Society for Biochemistry and Molecular Biology, Inc.

    09 Aug. 2013, Journal of Biological Chemistry, 288 (32), 23090 - 23104, English

    [Refereed]

    Scientific journal

  • Michal Bohdanowicz, Daniel Schlam, Martin Hermansson, David Rizzuti, Gregory D. Fairn, Takehiko Ueyama, Pentti Somerharju, Guangwei Du, Sergio Grinstein

    Macrophages and dendritic cells continuously survey their environment in search of foreign particles and soluble antigens. Such surveillance involves the ongoing extension of actin-rich protrusions and the consequent formation of phagosomes and macropinosomes. The signals inducing this constitutive cytoskeletal remodeling have not been defined. We report that, unlike nonphagocytic cells, macrophages and immature dendritic cells have elevated levels of phosphatidic acid (PA) in their plasma membrane. The plasmalemmal PA is synthesized by phosphorylation of diacylglycerol, which is in turn generated by a G protein-stimulated phospholipase C. Inhibition of diacylglycerol kinase activity results in the detachment of T-cell lymphoma invasion and metastasis-inducing protein 1 (TIAM1)-a Rac guanine exchange factor-from the plasma membrane, thereby depressing Rac activity and abolishing the constitutive ruffling and macropinocytosis that characterize macrophages and immature dendritic cells. Accumulation of PA and binding of TIAM1 to the membrane require the activity of phosphatidylinositol-4,5-bisphosphate 3-kinase. Thus a distinctive, constitutive pathway of PA biosynthesis promotes the actin remodeling required for immune surveillance. © 2013 Bohdanowicz et al.

    01 Jun. 2013, Molecular Biology of the Cell, 24 (11), 1700 - 1712, English

    [Refereed]

    Scientific journal

  • Kouhei Yamashita, Takashi Miyoshi, Yasuyuki Arai, Kiyomi Mizugishi, Akifumi Takaori-Kondo, Takehiko Ueyama

    Invasive pulmonary aspergillosis (IPA) is a life-threatening complication of chronic granulomatous disease (CGD), a rare inherited disorder of phagocytes that is characterized by a defect in the production of reactive oxygen species (ROS) caused by mutations in NADPH oxidase 2. Here, we report a case of successful treatment of IPA complicated with CGD by the administration of interferon-gamma (IFN-gamma) in combination with voriconazole. The patient carried a splice site mutation in the CYBB gene, and the neutrophils could produce a certain amount of ROS. In this case, augmentation of ROS generation in the patient's neutrophils was observed after in vivo IFN-gamma treatment, which may be attributable to the induction of a normal CYBB gene in the myeloid progenitor cells. This treatment, in combination with voriconazole, may have contributed to the reversal of IPA in this patient. These results suggest that the in vivo use of IFN-gamma may augment ROS generation in CGD neutrophils, thus leading to the successful treatment of severe IPA.

    SPRINGER JAPAN KK, Apr. 2013, INTERNATIONAL JOURNAL OF HEMATOLOGY, 97 (4), 505 - 510, English

    [Refereed]

    Scientific journal

  • Takehiko Ueyama, Junya Nakakita, Takashi Nakamura, Takeshi Kobayashi, Toshihiro Kobayashi, Jeonghyun Son, Megumi Sakuma, Hirofumi Sakaguchi, Thomas L. Leto, Naoaki Saito

    During activation of the phagocyte (Nox2-based) NADPH oxidase, the cytoplasmic Phox complex (p47(phox)-p67(phox) p40(phox) translocates and associates with the membrane-spanning flavocytochrome b(558). It is unclear where (in cytoplasm or on membranes), when (before or after assembly), and how p40(phox) acquires its PI(3)P-binding capabilities. We demonstrated that in addition to conformational changes induced by H2O2 in the cytoplasm, p40(phox) acquires PI(3)P-binding through direct or indirect membrane targeting. We also found that p40(phox) is essential when p47(phox) is partially phosphorylated during Fc gamma R-mediated oxidase activation; however, p40(phox) is less critical when p47(phox) is adequately phosphorylated, using phosphorylation-mimicking mutants in HEK293(Nox2/Fc gamma RIIa) and RAW264.7(P40/P47KD) cells. Moreover, PI binding to p47(phox) is less important when the autoinhibitory PX-PB1 domain interaction in p40(phox) is disrupted or when p40(phox) is targeted to membranes. Furthermore, we suggest that high affinity PI(3)P binding of the p40(phox) PX domain is critical during its accumulation on phagosomes, even when masked by the PB1 domain in the resting state. Thus, in addition to mechanisms for directly acquiring P I(3)P binding in the cytoplasm by H2O2, p40(phox) can acquire PI(3)P binding on targeted membranes in a p47(phox)-dependent manner and functions both as a "carrier" of the cytoplasmic Phox complex to phagosomes and an "adaptor" of oxidase assembly on phagosomes in cooperation with p47(phox), using positive feedback mechanisms.

    AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, Nov. 2011, JOURNAL OF BIOLOGICAL CHEMISTRY, 286 (47), 40693 - 40705, English

    [Refereed]

    Scientific journal

  • Yasuhito Shirai, Shoko Morioka, Megumi Sakuma, Ken-ichi Yoshino, Chihiro Otsuji, Norio Sakai, Kaori Kashiwagi, Kazuhiro Chida, Ryutaro Shirakawa, Hisanori Horiuchi, Chikako Nishigori, Takehiko Ueyama, Naoaki Saito

    During differentiation, keratinocytes undergo a dramatic shape change from small and round to large and flat, in addition to production of proteins necessary for the formation of epidermis. It has been shown that protein kinase C (PKC) eta is crucial for keratinocyte differentiation. However, its role in this process has yet to be fully elucidated. Here, we show that catalytic activity is not necessary for enlarged and flattened morphology of human keratinocytes induced by overexpression of PKC eta, although it is important for gene expression of the marker proteins. In addition, we identify the small G protein RalA as a binding partner of PKC eta, which binds to the C1 domain, an indispensable region for the morphological change. The binding led activation of RalA and actin depolymerization associated with keratinocyte differentiation. siRNA techniques proved that RalA is involved in not only the keratinocyte differentiation induced by PKC eta overexpression but also normal keratinocyte differentiation induced by calcium and cholesterol sulfate. These results provide a new insight into the molecular mechanism of cytoskeletal regulation leading to drastic change of cell shape.

    AMER SOC CELL BIOLOGY, Apr. 2011, MOLECULAR BIOLOGY OF THE CELL, 22 (8), 1340 - 1352, English

    [Refereed]

    Scientific journal

  • Takumi Kawasaki, Takehiko Ueyama, Ingo Lange, Stefan Feske, Naoaki Saito

    Ca2+ signals through store-operated Ca2+ (SOC) channels, activated by the depletion of Ca2+ from the endoplasmic reticulum, regulate various physiological events. Orai1 is the pore-forming subunit of the Ca2+ release-activated Ca2+ (CRAC) channel, the best characterized SOC channel. Orai1 is activated by stromal interaction molecule (STIM) 1, a Ca2+ sensor located in the endoplasmic reticulum. Orai1 and STIM1 are crucial for SOC channel activation, but the molecular mechanisms regulating Orai1 function are not fully understood. In this study, we demonstrate that protein kinase C (PKC) suppresses store-operated Ca2+ entry (SOCE) by phosphorylation of Orai1. PKC inhibitors and knockdown of PKC beta both resulted in increased Ca2+ influx. Orai1 is strongly phosphorylated by PKC in vitro and in vivo at N-terminal Ser-27 and Ser-30 residues. Consistent with these results, substitution of endogenous Orai1 with an Orai1 S27A/S30A mutant resulted in increased SOCE and CRAC channel currents. We propose that PKC suppresses SOCE and CRAC channel function by phosphorylation of Orai1 at N-terminal serine residues Ser-27 and Ser-30.

    AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, Aug. 2010, JOURNAL OF BIOLOGICAL CHEMISTRY, 285 (33), 25720 - 25730, English

    [Refereed]

    Scientific journal

  • Eisuke Adachi, Yutaka Kazoe, Yohei Sato, Yuko Suzuki, Tetsumei Urano, Takehiko Ueyama, Naoaki Saito, Viacheslav O. Nikolaev, Martin J. Lohse, Makoto Tominaga, Hideo Mogami

    Physiological phenomena are regulated by multiple signal pathways upon receptor stimulation. Here, we have introduced a new technique with a combination of prism-based total internal reflection fluorescence microscopy (PBTIRFM) and epifluorescence microscopy (EPI) to simultaneously monitor multiple signal pathways. This instrumentation allows us to visualize three signal pathways, Ca2+, cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA), and diacylglycerol (DAG)/protein kinase C (PKC) signals in living cells. Three fluorescent indicators were employed for this purpose: (1) Fura-2 AM as a calcium sensor; (2) Epac1-camp, a cyan fluorescent protein-yellow fluorescent protein fluorescence resonance energy transfer-based cAMP indicator, as a cAMP sensor; and (3) C1-tagged monomeric red fluorescent protein, a tandem DAG-binding domain of PKC gamma, as a DAG sensor or myristoylated alanine-rich C kinase substrate-tagged DsRed for the PKC activation pathway. The DAG signal was monitored by PBTIRFM, whereas the Ca2+ and cAMP signals were monitored by EPI. Adenosine trisphosphate resulted in generation of all three second messengers in triple probe-loaded Cos-7 cells. The spectral overlap between these signal probes was evaluated by means of linear unmixing. Forskolin also evoked Ca2+, cAMP/PKA, and DAG/PKC signals whereas acetylcholine activated Ca2+ and DAG/PKC signals as well as inhibiting cAMP generation in triple probe-loaded insulin-secreting cells. Thus, the optical observation system combining PBTIRFM and EPI offers a great advance in analyzing interplay of multiple signaling pathways, such as these second messengers, upon G-protein-coupled receptor stimulation in living cells.

    SPRINGER, Nov. 2009, PFLUGERS ARCHIV-EUROPEAN JOURNAL OF PHYSIOLOGY, 459 (1), 227 - 234, English

    [Refereed]

    Scientific journal

  • Thomas L. Leto, Stanislas Morand, Darrell Hurt, Takehiko Ueyama

    Nox family NADPH oxidases serve a variety of functions requiring reactive oxygen species (ROS) generation, including antimicrobial defense, biosynthetic processes, oxygen sensing, and redox-based cellular signaling. We explored targeting, assembly, and activation of several Nox family oxidases, since ROS production appears to be regulated both spatially and temporally. Nox1 and Nox3 are similar to the phagocytic (Nox2-based) oxidase, functioning as multicomponent superoxide-generating enzymes. Factors regulating their activities include cytosolic activator and organizer proteins and GTP-Rac. Their regulation varies, with the following rank order: Nox2>Nox1>Nox3. Determinants of subcellular targeting include: (a) formation of Nox-p22(phox) heterodimeric complexes allowing plasma membrane translocation, (b) phospholipids-binding specificities of PX domain-containing organizer proteins (p47(phox) or Nox organizer 1 (Noxo1 and p40(phox)), and (c) variably splicing of Noxo1 PX domains directing them to nuclear or plasma membranes. Dual oxidases (Duox1 and Duox2) are targeted by different mechanisms. Plasma membrane targeting results in H2O2 release, not superoxide, to support extracellular peroxidases. Human Duox1 and Duox2 have no demonstrable peroxidase activity, despite their extensive homology with heme peroxidases. The dual oxidases were reconstituted by Duox activator 2 (Duoxa2) or two Duoxa1 variants, which dictate maturation, subcellular localization, and the type of ROS generated by forming stable complexes with Duox. Antioxid Redox Signal. 11, 2607-2619.

    Last, MARY ANN LIEBERT, INC, Oct. 2009, ANTIOXIDANTS & REDOX SIGNALING, 11 (10), 2607 - 2619, English

    [Refereed]

    Scientific journal

  • Stanislas Morand, Takehiko Ueyama, Satoshi Tsujibe, Naoaki Saito, Agnieszka Korzeniowska, Thomas L. Leto

    Dual oxidases (Duox1 and Duox2) are plasma membrane-targeted hydrogen peroxide generators that support extracellular hemoperoxidases. Duox activator 2 (Duoxa2), initially described as an endoplasmic reticulum resident protein, functions as a maturation factor needed to deliver active Duox2 to the cell surface. However, less is known about the Duox1/Duoxa1 homologues. We identified four alternatively spliced Duoxa1 variants and explored their roles in Duox subcellular targeting and reconstitution. Duox1 and Duox2 are functionally rescued by Duoxa2 or the Duoxa1 variants that contain the third coding exon. All active maturation factors are cotransported to the cell surface when coexpressed with either Duox1 or Duox2, consistent with detection of endogenous Duoxa1 on apical plasma membranes of the airway epithelium. In contrast, the Duoxa proteins are retained in the endoplasmic reticulum when expressed without Duox. Duox1/Duoxa1 alpha and Duox2/Duoxa2 pairs produce the highest levels of hydrogen peroxide, as they undergo Golgi-based carbohydrate modifications and form stable cell surface complexes. Cross-functioning pairs that do not form stable complexes produce less hydrogen peroxide and leak superoxide. These findings suggest Duox activators not only promote Duox maturation, but they function as part of the hydrogen peroxide-generating enzyme.-Morand, S., Ueyama, T., Tsujibe, S., Saito, N., Korzeniowska, A., Leto, T. L. Duox maturation factors form cell surface complexes with Duox affecting the specificity of reactive oxygen species generation. FASEB J. 23, 1205-1218 (2009)

    FEDERATION AMER SOC EXP BIOL, Apr. 2009, FASEB JOURNAL, 23 (4), 1205 - 1218, English

    [Refereed]

    Scientific journal

  • Noriaki Ashida, Takehiko Ueyama, Kyoko Rikitake, Yasuhito Shirai, Mika Eto, Takeshi Kondoh, Eiji Kohmura, Naoaki Saito

    We found that stimulation of P2Y2 receptor (P2Y2R), which is endogenously expressed in CHO-K1 cells, induced intracellular calcium ([Ca2+](i)) Oscillation with a low frequency of 11.4 +/- 23 mHz. When CHO-K1 cells expressing GFP-tagged kinase-negative gamma PKC (gamma PKC-KN-GFP), which is a neuron-specific subtype of PKC, were stimulated with UDP, gamma PKC-KN-GFP, but not wild-type gamma PKC (gamma PKC-GFP) showed an oscillatory translocation. The oscillatory translocation of gamma PKC-KN-GFP corresponded with [Ca2+](i) oscillation, which was not observed in the cells expressing gamma PKC-GFP. We examined the mechanism of P2Y2R-induced [Ca2+](i) oscillation pharmacologically. gamma PKC-KN-GFP oscillation was stopped by an extracellular Ca2+ chelator, EGTA, an antagonist of P2Y2R, Suramin, and store-operated calcium channel (SOC) inhibitors, SKF96365 and 2-ABP. Taken together, P2Y2R-induced [Ca2+](i) oscillation in CHO-K1 cells is related with Ca2+ influx through SOC, whose function may be negatively regulated by gamma PKC. This [Ca2+](i), oscillation was distinct from that induced by metabotropic glutamate receptor 5 (mGluR5) stimulation in the frequency (72.3 +/- 5.3 mHz) and in the regulatory mechanism. (c) 2008 Elsevier Ireland Ltd. All rights reserved.

    ELSEVIER IRELAND LTD, Dec. 2008, NEUROSCIENCE LETTERS, 446 (2-3), 123 - 128, English

    [Refereed]

    Scientific journal

  • Sequential binding of cytosolic phox complex to phagosomes through regulated adaptor proteins: Evaluation using the novel monomeric kusabira-green system and live imaging of phagocytosis

    Takehiko Ueyama, Tomoko Kusakabe, Satoshi Karasawa, Takumi Kawasaki, Aya Shimizu, Jeonghyun Son, Thomas L. Leto, Atsushi Miyawaki, Naoaki Saito

    We engineered a method for detecting intramolecular and intermolecular phox protein interactions in cells by fluorescence microscopy using fusion proteins of complementary fragments of a coral fluorescent reporter protein (monomeric Kusabira-Green). We confirmed the efficacy of the monomeric Kusabira-Green system by showing that the PX and PB1 domains of p40(phox) interact in intact cells, which we suggested maintains this protein in an inactive closed conformation. Using this system, we also explored intramolecular interactions within p47(phox) and showed that the PX domain interacts with the autoinhibited tandem Src homology 3 domains maintained in contact with the autoinhibitory region, along with residues 341-360. Furthermore, we demonstrated sequential interactions of p67(phox) , With phagosomes involving adaptor proteins, p47(phox) and p40(phox), during Fc gamma R-mediated phagocytosis. Although p67(phox) is not targeted to phagosomes by itself, p47(phox) functions as an adaptor for the ternary complex (p47(phox), -p67(phox)-p40(phox)) in early stages of phagocytosis before phagosome closure, while p40(phox) functions in later stages after phagosomal closure. Interestingly, a mutated "open" form of p40(phox), linked p47(phox), to closed phagosomes and prolonged p47(phox) and p67(phox) retention on phagosomes. These results indicate that binding of the ternary complex to phagosomes can be temporally regulated by switching between adaptor proteins that have PX domains with distinct lipid-binding specificities.

    Lead, AMER ASSOC IMMUNOLOGISTS, Jul. 2008, JOURNAL OF IMMUNOLOGY, 181 (1), 629 - 640, English

    [Refereed]

    Scientific journal

  • Naoko Adachi, Takeshi Kobayashi, Hideyuki Takahashi, Takumi Kawasaki, Yasuhito Shirai, Takehiko Ueyama, Toshio Matsuda, Takahiro Seki, Norio Sakai, Naoaki Saito

    Spinocerebellar ataxia type 14 (SCA14) is an autosomal dominant neurodegenerative disease caused by mutations in protein kinase C gamma(PKC gamma). Interestingly, 18 of 22 mutations are concentrated in the C1 domain, which binds diacylglycerol and is necessary for translocation and regulation of PKC gamma kinase activity. To determine the effect of these mutations on PKC gamma function and the pathology of SCA14, we investigated the enzymological properties of the mutant PKC gamma s. We found that wild-type PKC gamma, but not C1 domain mutants, inhibits Ca2+ influx in response to muscarinic receptor stimulation. The sustained Ca2+ influx induced by muscarinic receptor ligation caused prolonged membrane localization of mutant PKC gamma. Pharmacological experiments showed that canonical transient receptor potential (TRPC) channels are responsible for the Ca2+ influx regulated by PKC gamma. Although in vitro kinase assays revealed that most C1 domain mutants are constitutively active, they could not phosphorylate TRPC3 channels in vivo. Single molecule observation by the total internal reflection fluorescence microscopy revealed that the membrane residence time of mutant PKC gamma s was significantly shorter than that of the wild-type. This fact indicated that, although membrane association of the C1 domain mutants was apparently prolonged, these mutants have a reduced ability to bind diacylglycerol and be retained on the plasma membrane. As a result, they fail to phosphorylate TRPC channels, resulting in sustained Ca2+ entry. Such an alteration in Ca2+ homeostasis and Ca2+-mediated signaling in Purkinje cells may contribute to the neurodegeneration characteristic of SCA14.

    AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, Jul. 2008, JOURNAL OF BIOLOGICAL CHEMISTRY, 283 (28), 19854 - 19863, English

    [Refereed]

    Scientific journal

  • Takumi Kawasaki, Takeshi Kobayashi, Takehiko Ueyama, Yasuhito Shirai, Naoaki Saito

    DGK delta (diacylglycerol kinase 3), which phosphorylates DAG (diacylglycerol) and converts it into PA (phosphatidic acid), has an important role in signal transduction. In the present study, we have demonstrated the molecular mechanism of DGK delta-mediated regulation of clathrin-dependent endocytosis that controls the internalization, recycling and degradation of receptors. Involvement of DGK6 in the regulation of clathrin-dependent endocytosis was previously proposed following genome-wide RNAi (RNA interference) screening. Clathrin-coated pits are mainly formed by clathrin and AP-2 (adaptor protein 2) complex. These proteins assemble a polyhedral lattice at the membrane and gather several endocytic accessory proteins. As the intracellular localization of DGK delta 2 overlapped with clathrin-coated pits, we predicted the possible regulation of clathrin-dependent endocytosis by DGK delta 2 and its interaction with some endocytosis-regulatory proteins. DGK delta 2 contained the DXF-type binding motifs, and DGK delta 2 bound to AP2 alpha, a subunit of the AP-2 complex. DGK delta 2 interacted with the platform subdomain in the AP2 alpha ear domain via (FDTFRIL)-D-369 and (DPF)-P-746 sequences in the catalytic domain of DGK delta 2. For further insight into the role for DGK delta 2 in clathrin-dependent endocytosis, we measured the transferrin and EGF (epidermal growth factor) uptakeexpressing wild-type or mutant DGK delta 2 under knockdown of endogenous DGK delta. Mutants lacking binding ability to AP2 alpha as well as kinase-negative mutants could not compensate for the uptake of transferrin inhibited by siRNA (small interfering RNA) treatment, whereas overexpression of wild-type DGK delta 2 completely recovered the transferrin uptake. These results demonstrate that binding between DGK delta 2 and AP2 alpha is involved in the transferrin internalization and that DGK activity is also necessary for the regulation of the endocytic process.

    PORTLAND PRESS LTD, Jan. 2008, BIOCHEMICAL JOURNAL, 409, 471 - 479, English

    [Refereed]

    Scientific journal

  • Takehiko Ueyama, Toshihiko Tatsuno, Takumi Kawasaki, Satoshi Tsujibe, Yasuhito Shirai, Hideki Sumimoto, Thomas L. Leto, Naoaki Saito

    In the phagocytic cell, NADPH oxidase (Nox2) system, cytoplasmic regulators (p47(phox), p67(phox), p40(phox), and Rac) translocate and associate with the membrane-spanning flavocytochrome b(558), leading to activation of superoxide production. We examined membrane targeting of phox proteins and explored conformational changes in p40(phox) that regulate its translocation to membranes upon stimulation. GFP-p40(phox) translocates to early endosomes, whereas GFP-p47(phox) translocates to the plasma membrane in response to arachidonic acid. In contrast, GFP-p67(phox) does not translocate to membranes when expressed alone, but it is dependent on p40(phox) and p47(phox) for its translocation to early endosomes or the plasma membrane, respectively. Translocation of GFP-p40(phox) or GFP-p47(phox) to their respective membrane-targeting sites is abolished by mutations in their phox (PX) domains that disrupt their interactions with their cognate phospholipid ligands. Furthermore, GFP-p67(phox) translocation to either membrane is abolished by mutations that disrupt its interaction with p40(phox) or p47(phox). Finally, we detected a head-to-tail (PX-Phox and Bem1 [PB1] domain) intramolecular interaction within p40(phox) in its resting state by deletion mutagenesis, cell localization, and binding experiments, suggesting that its PX domain is inaccessible to interact with phosphatidylinositol 3-phosphate without cell stimulation. Thus, both p40(phox) and p47(phox) function as diverse p67(phox) "carrier proteins" regulated by the unmasking of membrane-targeting domains in distinct mechanisms.

    Lead, AMER SOC CELL BIOLOGY, Feb. 2007, MOLECULAR BIOLOGY OF THE CELL, 18 (2), 441 - 454, English

    [Refereed]

    Scientific journal

  • Takehiko Ueyama, Kristen Lekstrom, Satoshi Tsujibe, Naoaki Saito, Thomas L. Leto

    Nox organizer 1 (Noxo1), a p47(phox) homolog, is produced as four isoforms with unique N-terminal PX domains derived by alternative mRNA splicing. We compared the subcellular distribution of these isoforms or their isolated PX domains produced as GFP fusion proteins, as well as their ability to support Nox1 activity in several transfected models. Noxo1 alpha, beta, gamma, and delta show different subcellular localization patterns, determined by their PX domains. In HEK293 cells, Noxo1 beta exhibits prominent plasma membrane binding, Noxo1 gamma shows plasma membrane and nuclear associations, and Noxo1 alpha and 6 localize primarily on intracellular vesicles or cytoplasmic aggregates, but not the plasma membrane. Nox1 activity correlates with Noxo1 plasma membrane binding in HEK293 cells, since Noxo1 beta supports the highest activity and Noxo1 gamma and Noxo1 alpha support moderate or low activities, respectively. In COS-7 cells, where Noxo1 alpha localizes on the plasma membrane, the activities supported by the three isoforms (alpha, beta, and gamma) do not differ significantly. The PX domains of beta and gamma bind the same phospholipids, including phosphatidic acid. These results indicate that the variant PX domains are unique determinants of Noxo1 localization and Nox1 function. Finally, the overexpressed Noxo1 isoforms do not affect p22(phox) localization, although Nox1 is needed to transport p22(phox) to the plasma membrane. Published by Elsevier Inc.

    ELSEVIER SCIENCE INC, Jan. 2007, FREE RADICAL BIOLOGY AND MEDICINE, 42 (2), 180 - 190, English

    [Refereed]

    Scientific journal

  • T Matsubara, Y Shirai, K Miyasaka, T Murakami, Y Yamaguchi, T Ueyama, M Kai, F Sakane, H Kanoh, T Hashimoto, S Kamada, U Kikkawa, N Saito

    Diacylglycerol kinases (DGKs) convert diacylglycerol (DG) to phosphatidic acid, and both lipids are known to play important roles in lipid signal transduction. Thereby, DGKs are considered to be a one of the key players in lipid signaling, but its physiological function remains to be solved. In an effort to investigate one of nine subtypes, we found that DGK gamma came to be localized in the nucleus with time in all cell lines tested while seen only in the cytoplasm at the early stage of culture, indicating that DGK gamma is transported from the cytoplasm to the nucleus. The nuclear transportation of DGK gamma didn't necessarily need DGK activity, but its C1 domain was indispensable, suggesting that the C1 domain of DGK gamma acts as a nuclear transport signal. Furthermore, to address the function of DGK gamma in the nucleus, we produced stable cell lines of wild-type DGK gamma and mutants, including kinase negative, and investigated their cell size, growth rate, and cell cycle. The cells expressing the kinase-negative mutant of DGK gamma were larger in size and showed slower growth rate, and the S phase of the cells was extended. These findings implicate that nuclear DGK gamma regulates cell cycle.

    AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, Mar. 2006, JOURNAL OF BIOLOGICAL CHEMISTRY, 281 (10), 6152 - 6164, English

    [Refereed]

    Scientific journal

  • T Ueyama, M Geiszt, TL Leto

    Several Nox family NADPH oxidases function as multicomponent enzyme systems. We explored determinants of assembly of the multicomponent oxidases Nox1 and Nox3 and examined the involvement of Rac1 in their regulation. Both enzymes are supported by p47(phox) and p67(phox) or homologous regulators called Noxo1 and Noxal, although Nox3 is less dependent on these cofactors for activity. Plasma membrane targeting of Noxal depends on Noxo1, through tail-to-tail interactions between these proteins. Noxal can support Nox1 without Noxo1, when targeted to the plasma membrane by fusing membrane-binding sequences from Rac1 (amino acids 183 to 192) to the C terminus of Noxal. However, membrane targeting of Noxal is not sufficient for activation of Nox1. Both the Noxo1-independent and -dependent Nox1 systems involve Rac1, since they are affected by Rac1 mutants or Noxal mutants defective in Rac binding or short interfering RNA-mediated Rac1 silencing. Nox1 or Nox3 expression promotes p22(phox) transport to the plasma membrane, and both oxidases are inhibited by mutations in the p22(phox) binding sites (SH3 domains) of the Nox organizers (p47(phox) or Noxo1). Regulation of Nox3 by Rac1 was also evident from the effects of mutant Rac1 or mutant Nox3 activators (p67(phox) or Noxa1) or Rac1 silencing. In the absence of Nox organizers, the Nox activators (p67(phox) or Noxa1) colocalize with Rac1 within ruffling membranes, independently of their ability to bind Rac1. Thus, Rac1 regulates both oxidases through the Nox activators, although it does not appear to direct the subcellular localization of these activators.

    AMER SOC MICROBIOLOGY, Mar. 2006, MOLECULAR AND CELLULAR BIOLOGY, 26 (6), 2160 - 2174, English

    [Refereed]

    Scientific journal

  • KL Cheeseman, T Ueyama, TM Michaud, K Kashiwagi, D Wang, LA Flax, Y Shirai, DJ Loegering, N Saito, MR Lennartz

    Protein kinase C-epsilon (PKC-epsilon) translocates to phagosomes and promotes uptake of IgG-opsonized targets. To identify the regions responsible for this concentration, green fluorescent protein (GFP)-protein kinase C-epsilon mutants were tracked during phagocytosis and in response to exogenous lipids. Deletion of the diacylglycerol (DAG)-binding epsilon C1 and epsilon C1B domains, or the epsilon C1B point mutant epsilon C259G, decreased accumulation at phagosomes and membrane translocation in response to exogenous DAG. Quantitation of GFP revealed that epsilon C259G, epsilon C1, and epsilon C1B accumulation at phagosomes was significantly less than that of intact PKC-epsilon. Also, the DAG antagonist 1-hexadecyl-2-acetyl glycerol (EI-150) blocked PKC-epsilon translocation. Thus, DAG binding to epsilon C1B is necessary for PKC-epsilon translocation. The role of phospholipase D (PLD), phosphatidylinositol-specific phospholipase C (PI-PLC)-gamma 1, and PI-PLC-gamma 2 in PKC-epsilon accumulation was assessed. Although GFP-PLD2 localized to phagosomes and enhanced phagocytosis, PLD inhibition did not alter target ingestion or PKC-epsilon localization. In contrast, the PI-PLC inhibitor U73122 decreased both phagocytosis and PKC-epsilon accumulation. Although expression of PI-PLC-gamma 2 is higher than that of PI-PLC-gamma 1, PI-PLC-gamma 1 but not PI-PLC-gamma 2 consistently concentrated at phagosomes. Macrophages from PI-PLC-gamma 2(-/-) mice were similar to wild-type macrophages in their rate and extent of phagocytosis, their accumulation of PKC-epsilon at the phagosome, and their sensitivity to U73122. This implicates PI-PLC-gamma 1 as the enzyme that supports PKC-epsilon localization and phagocytosis. That PI-PLC-gamma 1 was transiently tyrosine phosphorylated in nascent phagosomes is consistent with this conclusion. Together, these results support a model in which PI-PLC-gamma 1 provides DAG that binds to epsilon C1B, facilitating PKC-epsilon localization to phagosomes for efficient IgG-mediated phagocytosis.

    AMER SOC CELL BIOLOGY, Feb. 2006, MOLECULAR BIOLOGY OF THE CELL, 17 (2), 799 - 813, English

    [Refereed]

    Scientific journal

  • Isoform-specific membrane targeting mechanism of Rac during Fc gamma R-mediated phagocytosis: Positive charge-dependent and independent targeting mechanism of Rac to the phagosome

    T Ueyama, M Eto, K Kami, T Tatsuno, T Kobayashi, Y Shirai, MR Lennartz, R Takeya, H Sumimoto, N Saito

    Rac1 and Rac2 are capable of stimulating superoxide production in vitro, but their targeting and functional mechanisms are still unknown. In the present study, we found that Rac1, 2, and 3 all accumulate at the phagosome during Fc gamma R-mediated phagocytosis, and that the order of accumulation (Rac1 > Rac3 > Rac2) depends on the net positive charge in their polybasic (PB) regions (183-188 aa). Although all GFP-tagged prenylated PB regions of Rae isoforms (GFP-Rac(PB)) and GFP-tagged prenylated 6 Ala (GFP-6A) accumulated during phagocytosis, GFP-Rac2(PB) and GFP-6A showed weak accumulation at the phagosome through a linear structure connecting the phagosome and endomembranes. The PB region of Rac1 showed strong phospholipid interaction with PI(3)P, PI(4)P, PI(5)P, PI(3,4,5)P-3, and phosphatidic acid, however, that of Rac2 did not. Constitutively active Rac2, GFP-Rac2(Q61L), was predominantly localized at the endomembranes; these endomembranes fused to the phagosome through the linear structure during phagocytosis, and this accumulation mechanism did not depend on positive charge in the PB region. Our conclusion is that Rac1 directly targets to the phagosome using the positively charged PB region and this accumulation mechanism is likely enhanced by the phospholipids. In addition to this mechanism, Rac2 has a positive charge-independent mechanism in which Rac2 initially targets to endomembranes and then these endomembranes fuse to the phagosome.

    Lead, AMER ASSOC IMMUNOLOGISTS, Aug. 2005, JOURNAL OF IMMUNOLOGY, 175 (4), 2381 - 2390, English

    [Refereed]

    Scientific journal

  • S Hayashi, T Ueyama, T Kajimoto, K Yagi, E Kohmura, N Saito

    The neuroprotective effects of estrogen were studied in the ischemic model mice by 90 min transient unilateral middle cerebral artery occlusion (MCAO) followed by 22.5 h reperfusion. The total infarct size in C57BL/6 female mice after MCAO and reperfusion was significantly smaller than that in male mice. Intraperitoneal injection of estrogen after the start of reperfusion significantly reduced the infarct volume in the male mice. However, no significant gender difference was found in total infarct size in gamma protein kinase C (PKC)-knockout mice, suggesting that the neuroprotective effects of estrogen are due to the activation of a specific subtype of PKC, gamma PKC, a neuron-specific PKC subtype, in the brain. We demonstrated that exogenous estrogen-induced neuroprotection was attenuated in gamma PKC-knockout mice. Immunocytochemical study showed that gamma PKC was translocated to nerve fiber-like structures when observed shortly after MCAO and reperfusion. We also visualized the rapid and reversible translocation of gamma PKC-GFP (green fluorescent protein) by estrogen stimulation in living CHO-K1 cells. These results suggest that the activation of gamma PKC through the G-protein-coupled estrogen receptors on the plasma membrane is involved in the estrogen-induced neuroprotection against focal brain ischemia.

    BLACKWELL PUBLISHING LTD, May 2005, JOURNAL OF NEUROCHEMISTRY, 93 (4), 883 - 891, English

    [Refereed]

    Scientific journal

  • Superoxide production at phagosomal cup/phagosome through beta I protein kinase C during Fc gamma R-mediated phagocytosis in microglia

    T Ueyama, MR Lennartz, Y Noda, T Kobayashi, Y Shirai, K Kikitake, T Yamasaki, S Hayashi, N Sakai, H Seguchi, M Sawada, H Sumimoto, N Saito

    Protein kinase C (PKC) plays a prominent-role in immune signaling. To elucidate the signal transduction in a respiratory burst and isoform-specific function of PKC during FcgammaR-mediated phagocytosis, we used live, digital fluorescence imaging of mouse microglial cells expressing GFP-tagged molecules. betaI PKC, epsilonPKC, and diacylglycerol kinase (DGK) beta dynamically and transiently accumulated around IgG-opsonized beads (BIgG). Moreover, the accumulation of p47(phox), an essential cytosolic component of NADPH oxidase and a. substrate for PI PKC, at the phagosomal cup/phagosome was apparent during BIgG ingestion. Superoxide (O-2(-)) production was profoundly inhibited by Go6976, a cPKC inhibitor, and dramatically increased by the DGK inhibitor, R59949. Ultrastructural analysis revealed that BIgG induced O-2(-) production at the phagosome but not, at the intracellular granules. We conclude that activation/accumulation of betaI PKC is involved in O-2(-) production, and that O-2(-) production is primarily initiated at the phagosomal cup/phagosome. This study also suggests that DGKbeta plays a prominent role in regulation of O-2(-) production during FcgammaR-mediated phagocytosis.

    Lead, AMER ASSOC IMMUNOLOGISTS, Oct. 2004, JOURNAL OF IMMUNOLOGY, 173 (7), 4582 - 4589, English

    [Refereed]

    Scientific journal

  • A Kageyama, M Oka, T Okada, S Nakamura, T Ueyama, N Saito, VJ Hearing, M Ichihashi, C Nishigori

    The involvement of phospholipase D (PLD) in the regulation of melanogenesis was examined. Treatment of B16 mouse melanoma cells with 12-O-tetradecanoylphorbol-13- acetate (TPA) resulted in the activation of PLD and a decrease in melanin content. 1-Butanol, but not 2-butanol, completely blocked the TPA-induced inhibition of melanogenesis, suggesting the involvement of PLD in this event. Reverse transcription-PCR and immunoblot analyses revealed the existence of both PLD isozymes, PLD1 and PLD2, in B16 cells. When PLD1 or PLD2 was introduced into those cells by an adenoviral gene-transfer technique, both PLD1 and PLD2 were activated by TPA. When PLD1 and PLD2 were overexpressed, PLD2 potently caused a decrease in melanin content, whereas the effect of PLD1 expression on melanin content was minimal. Over-expression of PLD2 itself did not affect protein kinase C activity, as assessed by the intracellular distribution and levels of expression of each isoform expressed in B16 cells. The effects of TPA on the down-regulation of basal or alpha-melanocyte-stimulating hormone-enhanced melanogenesis were almost completely blocked by expressing a lipase activity-negative mutant, LN-PLD2, but not by LN-PLD1. Further, the PLD2-induced decrease in melanin content was accompanied by a decrease in the amount and activity of tyrosinase, a key enzyme in melanogenesis, whereas the mRNA level of tyrosinase was unchanged by the over-expression of PLD2. Moreover, treatment with proteasome inhibitors completely blocked the PLD2-induced down-regulation of melanogenesis. Taken together, the present results indicate that the TPA-induced down-regulation of melanogenesis is mediated by PLD2 but not by PLD1 through the ubiquitin proteasome-mediated degradation of tyrosinase. This suggests that PLD2 may play an important role in regulating pigmentation in vivo.

    AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, Jun. 2004, JOURNAL OF BIOLOGICAL CHEMISTRY, 279 (26), 27774 - 27780, English

    [Refereed]

    Scientific journal

  • AK Bhattacharjee, T Ueyama, T Kondoh, S Hayashi, A Abouelfetouh, N Sakai, N Saito, E Kohmura

    A recombinant adenoviral tetracycline-regulated system with neuron-specific enolase (NSE) promoter was injected stereotaxically into the striatum of rat brains. The efficiency of in vivo transfection was quantified by counting the number of green fluorescent protein (GFP)-positive cells at 3 days, 1 week, and 4 weeks after injection. NeuN immunohistochemistry demonstrated that expression of gammaPKC-GFP was dominant (20-99%) in neuron and expression of gammaPKC-GFP in neuron was significantly higher in pups than adult rats. These results indicate that tetracycline-inhibitable transcription factor (tTA) can drive tetracycline-responsive promoter (TetOp) under the control of NSE promoter, thereby efficiently and selectively expressing gammaPKC-GFP in neurons in vivo. (C) 2004 Elsevier Inc. All rights reserved.

    ACADEMIC PRESS INC ELSEVIER SCIENCE, May 2004, BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 317 (4), 1144 - 1148, English

    [Refereed]

    Scientific journal

  • K Sakai, C Hasegawa, M Okura, O Morikawa, T Ueyama, Y Shirai, N Sakai, N Saito

    Three variants of murine serotonin transporter (5-HTT) mRNA, which consist of a different exon-one (exon 1a, exon 1b or exon 1c) and the same exon-two to exon-five, were identified. The promoter region for each exon 1 (p1a, p1b and p1c, respectively), ligated to pGL-3 enhancer vector, had activities significantly higher than the empty vector in all cell lines tested except p1c in PC-12, whereas the activity of p1c was significantly lower than the others. Effects of the treatment of dibutyryl-cyclic AMP, human interferon-a or mouse interferon-gamma have different profiles among COS-7, PC-12, C6 glioma and immortalized rat serotonergic raphe neurons, RN46A. These three promoter regions may play a role in the transcription of the 5-HTT and could offer a model of the regulation of 5-HTT production in humans and further the pathogenesis of depression. (C) 2003 Elsevier Science Ireland Ltd. All rights reserved.

    ELSEVIER SCI IRELAND LTD, May 2003, NEUROSCIENCE LETTERS, 342 (3), 175 - 178, English

    [Refereed]

    Scientific journal

  • A role for PKC-e in FcgR-mediated phagocytosis by RAW 264.7 cells

    Larsen EC, UEYAMA TAKEHIKO, Saito N, Lennartz MR

    Nov. 2002, J. Cell Biology, 159, 291 - 301, English

    [Refereed]

    Scientific journal

  • Naoki Irie, Norio Sakai, Takehiko Ueyama, Taketoshi Kajimoto, Yasuhito Shirai, Naoaki Saito

    RNA interference (RNAi), the targeted mRNA degradation induced by double-stranded RNA (dsRNA), is a powerful tool for analyzing gene function in many organisms. Recently, it has been shown that RNAi is also applicable to cultured mammalian cells by using short interfering RNA (siRNA) [Nature 411 (2001) 494]. To examine whether this siRNA method is useful for analyzing the subtype-specific functions of protein kinase C (PKC), we first prepared siRNAs which target human αPKC and human δPKC and applied them into mammalian cells to suppress the expression of endogenous αPKC and δPKC, respectively. Each siRNA for α or δPKC specifically suppressed the endogenous expression of corresponding PKC subtype in human-derived cell lines such as HEK-293 and HeLa cells, but not in cells derived from rat species. The suppression level of δPKC reached maximum 48-72 h after the transfection of siRNA. In addition, the siRNA targeting rat δPKC suppressed endogenous and exogenous rat δPKCs but not human δPKC, suggesting that siRNAs targeting PKCs effectively knocked down endogenous/exogenous PKCs in mammalian cells, in subtype- and species-specific manner. Furthermore, we also developed the method to discriminate the siRNA-transfected cells using the antibody recognizing thymine dimer. Our present results strongly suggest that siRNA method enable us to examine the subtype-specific function of PKC, not only by knockdown of the endogenous target PKC subtype, but also by subsequent compensation with the exogenous corresponding wild/mutant PKC derived from other species. © 2002 Elsevier Science (USA). All rights reserved.

    2002, Biochemical and Biophysical Research Communications, 298 (5), 738 - 743, English

    [Refereed]

    Scientific journal

  • Generation of a constitutively active fragment of PKN in microglia/macrophages after middle cerebral artery occlusion in rats

    Ueyama T, Y Ren, N Sakai, M Takahashi, Y Ono, T Kondoh, N Tamaki, N Saito

    PKN is a fatty acid- and Rho-activated serine/threonine kinase, which has a catalytic domain highly homologous to that of protein kinase C (PKC). Recent studies have demonstrated that PKN is proteolytically cleaved after apoptotic stimulation and then a constitutively active 55-kDa fragment is generated. However, the role of the 55-kDa fragment are poorly understood. Adult Sprague-Dawley (SD) rats underwent middle cerebral artery occlusion (MCAO), and the temporal and spatial changes in the fragmentation of PKN and of PKC delta were examined by immunoblotting. No proteolytic fragment of PKC delta (about 40 kDa) was detected. The 55-kDa fragment of PKN appeared transiently from 3 days after MCAO at the ipsilateral normal cortex. At the boundary zone of infarction, the 55-kDa fragment was markedly induced from day 5 then peaked on day 21 and persisted until day 28. Analysis of anti-phosphoserine immunoprecipitates with an anti-PKN antibody revealed phosphorylation of the 55-kDa band. Double staining for PKN and Ox42 was used to examine the source of the 55-kDa fragment. PKN immunoreactivity was significantly increased in Ox42-positive cells (microglia/hematogenous macrophages). No DNA laddering and only a few terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL)positive cells were observed on day 14 in despite of the high level appearance of the 55-kDa band. These results suggest that the constitutively active 55-kDa fragment of PKN does not contribute to apoptosis, but may contribute to a function of microglia/macrophages.

    Lead, BLACKWELL SCIENCE LTD, Nov. 2001, JOURNAL OF NEUROCHEMISTRY, 79 (4), 903 - 913, English

    [Refereed]

    Scientific journal

  • cDNA cloning of an alternative splicing variant of protein kinase C delta (PKC delta III), a new truncated form of PKC delta, in rats

    T Ueyama, Y Ren, S Ohmori, K Sakai, N Tamaki, N Saito

    Recently, an alternative splicing variant of mouse protein kinase C delta (PKC delta II, GenBank Accession No. AB011812) has been reported which has a 78 bp (26 amino acid) insertion at the caspase-3 recognition sequence in the V3 region of PKC delta (PKC delta I). We isolated a cDNA encoding a new variant of PKC delta (PKC delta III, AF219629), which has a 83 bp insertion at the same site in the V3 region, by RT-PCR using rat testis RNA as a template. In rats, the 83 bp insertion causes inframe termination, and rat PKC delta III protein is expressed as a truncated form, having only the regulatory domain without a catalytic domain. Genomic DNA analysis revealed that the difference between mouse PKC delta II and rat PKC delta III is derived from the different sequence at the 5'-splicing donor sites. To investigate the potential functions of the truncated form of PKC delta, rat PKC delta III fused to green fluorescent protein (GFP) was expressed in CHO-K1 cells. PKC delta III-GFP was localized in the cytoplasm with dot-like accumulation and highly expressed on the plasma membrane, whereas PKC delta I-GFP is localized homogeneously throughout the cytoplasm, including the nucleoplasm. Stimulation by phorbol ester caused weak translocation of delta III-GFP from the cytosol to the plasma membrane. These results suggest that PKC delta III may show a dominant negative effect against PKC delta I, and that the modulation of signal transduction by alternative splicing variant may play a crucial role in the physiological and/or pathological conditions, and the pathogenesis of disease. (C) 2000 Academic Press.

    Lead, ACADEMIC PRESS INC, Mar. 2000, BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 269 (2), 557 - 563, English

    [Refereed]

    Scientific journal

  • Non-traumatic acute paraplegia associated with cervical disk herniation: A case report

    UEYAMA TAKEHIKO

    Jan. 1999, Surgical Neurology, 52, 204 - 207, English

    [Refereed]

    Scientific journal

  • Large and invasive silent corticotroph-cell adenoma with elevated serum ACTH: A case report

    UEYAMA T, TAMAKI N, KONDOH T, KURATA H

    Jan. 1998, SURGICAL NEUROLOGY, 50 (1), 30 - 32, English

    [Refereed]

    Scientific journal

  • Cerebellopontine angle ependymoma with internal auditory canal enlargement and pineal extension

    Takehiko Ueyama, Norihiko Tamaki, Takeshi Kondoh, Takashi Kokunai, Masahiro Asada

    A 38-year-old male presented with a posterior fossa ependymoma with unusual extension from the cerebellopontine angle to the pineal region. Magnetic resonance imaging clearly demonstrated that these two components were continuous through the right ambient cistern. Computed tomography using a bone algorithm revealed enlargement of the right internal auditory canal. This case suggests that ependymoma can extend anywhere within the subarachnoid space along the path of least resistance.

    Oct. 1997, Neurologia Medico-Chirurgica, 37 (10), 762 - 765, English

    [Refereed]

    Scientific journal

  • Transsphenoidal surgery for a case of empty sella syndrome associated with GH secreting pituitary adenoma

    Hideo Aihara, Norihiko Tamaki, Takehiko Ueyama, Yousuke Ishihara, Takeshi Kondoh

    A 33-year-old woman was admitted to our hospital with acromegalic face as her chief complaint. Her neurological examination was normal, and endocrinological examination revealed a high level of growth hormone (GH) (12.8ng/ml). CT cisternography and MRI showed an enlarged empty sella and a pituitary tumor. We performed a transsphenoidal approach operation to remove the tumor and to repair the empty sella. The tumor, which was compressed to the lateral and posterior wall of the sella turcica by the empty sella, was totally removed by meticulous curetting. It was histologically diagnosed to be a pituitary adenoma. The empty sella was elevated by coagulation of intrasellar dura and herniated arachnoid membrane, and then we filled the residual intrasellar cavity with bone fragments and fat. Postoperative hormonal examination showed normal findings, and MRI revealed obliteration of the empty sella. Surgical indication for primary empty sella is not established, but in cases associated with pituitary adenoma, transsphenoidal surgery is necessary. We reported a case of empty sella syndrome associated with GH secreting pituitary adenoma, and in this report, we introduced a new surgical technique for repairing an empty sella.

    Dec. 1996, Neurological Surgery, 24 (12), 1119 - 1123, Japanese

    [Refereed]

    Scientific journal

  • Hiroaki Mohri, Yuzuru Ninoyu, Hirofumi Sakaguchi, Shigeru Hirano, Naoaki Saito, Takehiko Ueyama

    Reactive oxygen species (ROS) produced by NADPH oxidases (Nox) contribute to the development of different types of sensorineural hearing loss (SNHL), a common impairment in humans with no established treatment. Although the essential role of Nox3 in otoconia biosynthesis and its possible involvement in hearing have been reported in rodents, immunohistological methods targeted at detecting Nox3 expression in inner ear cells reveal ambiguous results. Therefore, the mechanism underlying Nox3-dependent SNHL remains unclear and warrants further investigation. We generated Nox3-Cre knock-in mice, in which Nox3 was replaced with Cre recombinase (Cre). Using Nox3-Cre;tdTomato mice of either sex, in which tdTomato is expressed under the control of the Nox3 promoter, we determined Nox3-expressing regions and cell types in the inner ear. Nox3-expressing cells in the cochlea included various types of supporting cells, outer hair cells, inner hair cells, and spiral ganglion neurons. Nox3 expression increased with cisplatin, age, and noise insults. Moreover, increased Nox3 expression in supporting cells and outer hair cells, especially at the basal turn of the cochlea, played essential roles in ROS-related SNHL. The extent of Nox3 involvement in SNHL follows the following order: cisplatin-induced hearing loss > age-related hearing loss > noise-induced hearing loss. Here, on the basis of Nox3-Cre;tdTomato, which can be used as a reporter system (Nox3-Cre +/- ;tdTomato +/+ and Nox3-Cre +/+ ;tdTomato +/+), and Nox3-KO (Nox3-Cre +/+ ;tdTomato +/+) mice, we demonstrate that Nox3 inhibition in the cochlea is a promising strategy for ROS-related SNHL, such as cisplatin-induced HL, age-related HL, and noise-induced HL.SIGNIFICANCE STATEMENT We found Nox3-expressing regions and cell types in the inner ear, especially in the cochlea, using Nox3-Cre;tdTomato mice, a reporter system generated in this study. Nox3 expression increased with cisplatin, age, and noise insults in specific cell types in the cochlea and resulted in the loss (apoptosis) of outer hair cells. Thus, Nox3 might serve as a molecular target for the development of therapeutics for sensorineural hearing loss, particularly cisplatin-induced, age-related, and noise-induced hearing loss.

    Corresponding, 26 May 2021, The Journal of neuroscience : the official journal of the Society for Neuroscience, 41 (21), 4716 - 4731, English, International magazine

    [Refereed]

    Scientific journal

  • Takehiko Ueyama

    The small GTPases of the Rho-family (Rho-family GTPases) have various physiological functions, including cytoskeletal regulation, cell polarity establishment, cell proliferation and motility, transcription, reactive oxygen species (ROS) production, and tumorigenesis. A relatively large number of downstream targets of Rho-family GTPases have been reported for in vitro studies. However, only a small number of signal pathways have been established at the in vivo level. Cumulative evidence for the functions of Rho-family GTPases has been reported for in vivo studies using genetically engineered mouse models. It was based on different cell- and tissue-specific conditional genes targeting mice. In this review, we introduce recent advances in in vivo studies, including human patient trials on Rho-family GTPases, focusing on highly polarized sensory organs, such as the cochlea, which is the primary hearing organ, host defenses involving reactive oxygen species (ROS) production, and tumorigenesis (especially associated with RAC, novel RAC1-GSPT1 signaling, RHOA, and RHOBTB2).

    28 Jan. 2019, Cells, 8 (2), English, International magazine

    [Refereed]

    Scientific journal

  • Bridging veins on the tentorial surface of the cerebellum: A microsurgical anatomic study and operative considerations

    Ueyama T, Al-Mefty O, Tamaki N

    Nov. 1998, Neurosurgery, 43 (5), 1137 - 1145

    [Refereed]

  • 難聴を生み出す活性酸素発生源細胞の同定とその機能・障害メカニズムの解明

    毛利 宏明, 二之湯 弦, 坂口 博史, 齋藤 尚亮, 上山 健彦

    日本組織細胞化学会, Sep. 2019, 日本組織細胞化学会総会・学術集会講演プログラム・予稿集, 60回, 74 - 74, Japanese

  • DFNA1疾患マウスモデルを用いたDIA1分子の局在と病態メカニズムの解明

    二之湯 弦, 坂口 博史, 陳 林, 毛利 宏明, 齋藤 尚亮, 上山 健彦

    日本組織細胞化学会, Sep. 2019, 日本組織細胞化学会総会・学術集会講演プログラム・予稿集, 60回, 83 - 83, Japanese

  • 活性酸素種(ROS)過剰発現モデルマウスにおける聴覚機能解析と聴覚保護因子の検索

    森岡 繁文, 坂口 博史, 山口 太郎, 二之湯 弦, 毛利 宏明, 中村 高志, 久 育男, 荻田 喜代一, 齋藤 尚亮, 上山 健彦

    日本組織細胞化学会, Sep. 2019, 日本組織細胞化学会総会・学術集会講演プログラム・予稿集, 60回, 84 - 84, Japanese

  • 坂口 博史, 二之湯 弦, 中村 高志, 森岡 繁文, 平野 滋, 上山 健彦, 齋藤 尚亮

    (一社)日本耳科学会, Sep. 2016, Otology Japan, 26 (4), 370 - 370, Japanese

  • PKCγKOパーキンソン症候群モデル βPIXリン酸化のドパミン遊離での役割

    白藤 俊彦, 上山 健彦, 吉野 健一, 足立 直子, 高橋 英之, 平松 直樹, 吾郷 由希夫, 松田 敏夫, 戸田 達史, 酒井 規雄, 齋藤 尚亮

    (一社)日本神経学会, Dec. 2014, 臨床神経学, 54 (Suppl.), S242 - S242, Japanese

  • 坂口 博史, 中村 高志, 森岡 繁文, 二之湯 弦, 上山 健彦, 齋藤 尚亮, 久 育男

    (一社)日本耳鼻咽喉科学会, Apr. 2014, 日本耳鼻咽喉科学会会報, 117 (4), 431 - 431, Japanese

  • 黒質線条体系におけるPKCγの基質の解析

    白藤 俊彦, 上山 健彦, 吉野 健一, 足立 直子, 高橋 英之, 香田 健, 梨子田 哲明, 吾郷 由希夫, 松田 敏夫, 齋藤 尚亮

    (公社)日本薬理学会, Oct. 2012, 日本薬理学雑誌, 140 (4), 1P - 1P, Japanese

  • Cysteine String Protein alpha(CSPα)Ser34特異的リン酸化抗体の作製と機能解析

    白藤 俊彦, 上山 健彦, 足立 直子, 齋藤 尚亮

    日本組織細胞化学会, Sep. 2019, 日本組織細胞化学会総会・学術集会講演プログラム・予稿集, 60回, 85 - 85, Japanese

MISC

  • A protein kinase C-gamma knockout Parkinsonian model: Involvement of Pak-Interacting Exchange Factor-beta

    Toshihiko Shirafuji, Takehiko Ueyama, Ken-ichi Yoshino, Naoko Adachi, Hideyuki Takahashi, Naoki Hiramatsu, Yukio Ago, Toshio Matsuda, Norio Sakai, Naoaki Saito

    JAPANESE PHARMACOLOGICAL SOC, 2014, JOURNAL OF PHARMACOLOGICAL SCIENCES, 124, 207P - 207P, English

    Summary international conference

  • Inner ear hair cell-specific Cdc42-KO mice demonstrate progressive hearing loss by impaired actin turnover in stereocilia and apical junctions

    Takashi Nakamura, Takehiko Ueyama, Hirofumi Sakaguchi, Akihiro Goto, Shigefumi Morioka, Shigenobu Yonemura, Yasuo Hisa, Michiyuki Matsuda, Atsu Aiba, Naoaki Satio

    JAPANESE PHARMACOLOGICAL SOC, 2014, JOURNAL OF PHARMACOLOGICAL SCIENCES, 124, 92P - 92P, English

    Summary international conference

  • Analysis of PKCY substrates in nigro-striatum system:The role of beta PIX phosphorylation in the dopamine release

    Toshihiko Shirafuji, Takehiko Ueyama, Kenichi Yoshino, Naoko Adachi, Hideyuki Takahashi, Yukio Ago, Toshio Matsuda, Naoaki Saito

    JAPANESE PHARMACOLOGICAL SOC, 2013, JOURNAL OF PHARMACOLOGICAL SCIENCES, 121, 122P - 122P, English

    Summary international conference

  • アストロサイト特異的Rac1ノックアウト(KO)による神経損傷後の機能回復促進

    ISHII TAIJI, KAMIYAMA TAKEHIKO, SAKAMOTO ISAO, SHIGYO MICHIKO, KUBOYAMA TOMOHARU, KOTA MASAAKI, TODA CHIHIRO, KOMURA EIJI, SAITO NAOAKI

    2013, 日本薬理学会近畿部会プログラム・要旨集, 124th, 31, Japanese

  • CNS injury and Rho family GTPases

    Takehiko Ueyama, Naoaki Saito

    JAPANESE PHARMACOLOGICAL SOC, 2012, JOURNAL OF PHARMACOLOGICAL SCIENCES, 118, 31P - 31P, English

    Summary international conference

  • The role of diacylglycerol kinase in histamine release

    Megumi Sakuma, Yasuhito Shirai, Takehiko Ueyama, Naoaki Saito

    JAPANESE PHARMACOLOGICAL SOC, 2012, JOURNAL OF PHARMACOLOGICAL SCIENCES, 118, 255P - 255P, English

    Summary international conference

  • Roles of Rho family GTPases during cerebellar development

    Masakazu Hayashibe, Takehiko Ueyama, Takeshi Kondoh, Jeonghyun Son, Mizuho Sakahara, Hidetoshi Kassai, Kazuki Nakao, Atsu Aiba, Naoaki Saito

    JAPANESE PHARMACOLOGICAL SOC, 2011, JOURNAL OF PHARMACOLOGICAL SCIENCES, 115, 113P - 113P, English

    Summary international conference

  • Regulatory mechanisms of Rac activation by RhoGDI

    Takehiko Ueyama, Jeonghyun Son, Takeshi Kobayashi, Naoaki Saito

    JAPANESE PHARMACOLOGICAL SOC, 2011, JOURNAL OF PHARMACOLOGICAL SCIENCES, 115, 29P - 29P, English

    Summary international conference

  • The function of P40(phox) in Nox2 activation

    Jun-ya Nakakita, Takehiko Ueyama, Toshihiro Kobayashi, Takeshi Kobayashi, Jon-hyun Son, Mio Nakatsuji, Yasuhito Shirai, Naoaki Saito

    JAPANESE PHARMACOLOGICAL SOC, 2010, JOURNAL OF PHARMACOLOGICAL SCIENCES, 112, 118P - 118P, English

    Summary international conference

  • The compensable function of Rac3 for Rac1 in mouse primary keratinocytes

    Aya Shimizu, Takehiko Ueyama, Mizuho Sakahara, Atsu Aiba, Naoaki Saito

    JAPANESE PHARMACOLOGICAL SOC, 2009, JOURNAL OF PHARMACOLOGICAL SCIENCES, 109, 71P - 71P, English

    Summary international conference

  • Differential regulation of Dual oxidase 1 by Duox activator 1 splicing variants

    Satoshi Tsujibe, Takehiko Ueyama, Yasuhito Shirai, Leto Thomas, Naoaki Saito

    JAPANESE PHARMACOLOGICAL SOC, 2008, JOURNAL OF PHARMACOLOGICAL SCIENCES, 106, 53P - 53P, English

    Summary international conference

  • Sequential targeting of cytosolic phox proteins to phagosomes through regulated adaptor proteins during Fc gamma R-mediated phagocytosis

    Takehiko Ueyama, Tomoko Kusakabe, Satoshi Karasawa, Takumi Kawasaki, Aya Shimizu, Jeong-hyun Son, Leto Thomas, Atsushi Miyawaki, Naoaki Saito

    JAPANESE PHARMACOLOGICAL SOC, 2008, JOURNAL OF PHARMACOLOGICAL SCIENCES, 106, 102P - 102P, English

    Summary international conference

  • PKC induced phosphorylation of Orai1 regulates intracellular Ca2+ level from store operated channel

    Takumi Kawasaki, Takehiko Ueyama, Yasuhito Shirai, Naoaki Saito

    JAPANESE PHARMACOLOGICAL SOC, 2008, JOURNAL OF PHARMACOLOGICAL SCIENCES, 106, 116P - 116P, English

    Summary international conference

  • Analysis of inter- and intra-molecular interactions of phox proteins using a new fluorescent protein at cellular levels

    Tomoko Kusakabe, Aya Shimizu, Takehiko Ueyama, Takumi Kawasaki, Yasuhito Shirai, Satosi Karasawa, Atsusi Miyawaki, Naoaki Saito

    JAPANESE PHARMACOLOGICAL SOC, 2007, JOURNAL OF PHARMACOLOGICAL SCIENCES, 103, 54P - 54P, English

    Summary international conference

  • A regulated adaptor function of p40phox: a head-to-tail (PX-PB1 domain) intramolecular interaction of p40phox in its resting state

    Takehiko Ueyama, Toshihiko Tatsuno, Takumi Kawasaki, Satosi Tsujibe, Yasuhito Shirai, Hideki Sumimoto, Thomas. L. Leto, Naoaki Saito

    JAPANESE PHARMACOLOGICAL SOC, 2007, JOURNAL OF PHARMACOLOGICAL SCIENCES, 103, 95P - 95P, English

    Summary international conference

Presentations

  • 生体防御に関する活性酸素産生酵素の食胞・頂側膜へのターゲティング及び会合メカニズム

    Ueyama Takehiko, Saito Naoaki

    第92回日本生理学会大会, Mar. 2015, Japanese, 日本生理学会, 神戸, Domestic conference

    [Invited]

    Invited oral presentation

  • Radial migration of cerebellar granule neuron regulated by Rac through a new signaling pathway.

    Ninoyu, Y, Ueyama Takehiko, Nakamura, T, Ishii, T, Kota Masaaki, Kasahara, M, Sakaguchi, H, Hisa, Y, Kohmura Eiji, Aiba, A, Saito Naoaki

    第88回日本薬理学会, Mar. 2015, Japanese, 日本薬理学会, 名古屋, Domestic conference

    Oral presentation

  • 内耳有毛細胞におけるRhoファミリー低分子量G蛋白質の役割

    Ueyama Takehiko, Saito Naoaki

    第55回日本組織細胞化学会, Sep. 2014, Japanese, 日本組織細胞化学会, 松本, Domestic conference

    Oral presentation

  • PKC KO Parkinsonian syndrome model:The role of PIX phosphorylation at Ser340 and Ser583 in dopamine release.

    Shirafuji, T, Ueyama Takehiko, Yoshino Kenichi, Adachi Naoko, Takahashi Harumi, Hiramatsu, N, Ago, Y, Matsuda, T, Toda Tatsushi, Sakai, N, Saito Naoaki

    第37回日本神経科学大会, Sep. 2014, Japanese, 日本神経科学会, 横浜, Domestic conference

    Poster presentation

  • Maintenance of stereocillia and apical junctional complexes by Cdc42 in cochlear hair cells.

    Ueyama Takehiko, Sakaguchi, H, Nakamura, T, Goto, A, Morioka, S, Fritzsch, B, Hisa, Y, Matsuda, M, Aiba, A, Saito Naoaki

    Society for Neuroscience 2014, Sep. 2014, English, Society for Neuroscience, Washington D.C., USA, International conference

    Oral presentation

  • Analysis of PKC substrates in nigro-striatum system:The role of bPIX phosphorylation for dopamine release.

    Shirafuji, T, Ueyama Takehiko, Yoshino Kenichi, Adachi Naoko, Takahashi Harumi, Hiramatsu, N, Ago, Y, Matsuda, T, Toda Tatsushi, Sakai, N, Saito Naoaki

    Society for Neuroscience 2014, Sep. 2014, English, Society for Neuroscience, Washington D.C., USA, International conference

    Oral presentation

  • 活性酸素産生NADPH oxidasesの食胞・頂側膜へのターゲティング及び会合メカニズム

    Ueyama Takehiko

    Asia International Institute of Infectious Disease Control, Aug. 2014, Japanese, Asia International Institute of Infectious Disease Control, 東京, Domestic conference

    [Invited]

    Invited oral presentation

  • Cdc42 is required for maintenance of stereocilia and apical junctional complexes in cochlear hair cells.

    Ueyama Takehiko, Sakaguchi, H, Goto, A, Hisa, Y, Matsuda, M, Aiba, A, Saito Naoaki

    The 37th Naito Conference, Jul. 2014, English, 内藤記念科学振興財団, 北海道, International conference

    Poster presentation

  • PKCノックアウトパーキンソン症候群モデル:bPIXリン酸化のドパミン遊離での役割

    白藤俊彦, Ueyama Takehiko, Yoshino Kenichi, Adachi Naoko, 高橋英之, 香田 健, 平松直樹, 吾郷 由希夫, 松田敏夫, Toda Tatsushi, 酒井規雄, Saito Naoaki

    第55回日本神経学会学術大会, May 2014, Japanese, 日本神経学会, 福岡, Domestic conference

    Poster presentation

  • アストロサイト特異的 Rac1 ノックアウト(KO)による神経損傷後の機能回復促進

    石井 大嗣, Ueyama Takehiko, 坂本 勲勇, 執行 美智子, 久保山 友晴, Kota Masaaki, 東田 千尋, Kohmura Eiji, Saito Naoaki

    第124回日本薬理学会近畿部会, Nov. 2013, Japanese, The Japanese Pharmacological Society, 京都, 【背景】Rhoファミリー低分子量G蛋白質であるRacは、細胞分裂・周期や細胞遊走などに関与することが知られている。一方、アストロサイトが中枢神経回路網の発生誘導・支持などに機能することは周知の事実であるが、神経損傷後の機能は複雑で、回復促進及び回復阻害のいずれの作用も報告がなされている。【方法】アストロサイト特異的Rac1 KOマウスを作製し、SPring8による微小放射線脳損傷及び圧挫による脊髄損傷を作製し、野生型マウスとの比較検討を行なった。【結果】小脳顆粒細胞に対する微小放射線脳損傷の検討では、 Rac1 KOマウスの損傷周囲のアストロサイトのGFAP染色性は低下し、小脳顆粒細胞の脱落幅も縮小していた(15.6μm vs 18.5μm)。BMS(Basso Mouse Scale)スコアを用いた脊髄損傷後の運動機能評価では、Rac1 KOマウス, Domestic conference

    Oral presentation

  • 神経損傷後の機能回復には低分子量G蛋白質Rac1を介したアストロサイト制御が関与する

    Kota Masaaki, Ueyama Takehiko, 執行 美智子, 近藤 威, 久保山 友晴, 石井 大嗣, 東田 千尋, Saito Naoaki, Kohmura Eiji

    The 72nd Annual Meeting of the Japan Neurosurgical Society, Oct. 2013, Japanese, Annual Meeting of the Japan Neurosurgical Society, 横浜, 【背景】Rhoファミリー低分子量Gタンパク質の1つであるRacは、細胞極性決定、細胞遊走など様々な機能に関与しているが、不明な点も多い。一方、アストロサイトは中枢神経回路網の支持、発生誘導などの働きを有するが、神経損傷後の機能は複雑で、回復促進および回復阻害のいずれの作用も報告されている。本研究では、アストロサイト特異的Rac1ノックアウト (KO) マウスを作製し、神経損傷後のアストロサイトの機能について検討した。尚、我々は以前から高輝度放射光施設(SPring-8)のマイクロビームX線照射による微小脳損傷モデルを用いた検討を行っており、本研究においても併用した。【方法】アストロサイト特異的Rac1 KOマウスに対して、微小脳損傷および脊髄損傷を作成し、野生型マウスとの比較検討をおこなった。【結果】小脳に対する微小脳損傷の検討では、小脳顆粒細胞の脱, Domestic conference

    Poster presentation

  • Involvment of PKCgamma in neurodegenerative disease

    Saito Naoaki, Takahashi Harumi, Shirafuji T, Adachi Naoko, Danno S, Ueyama Takehiko, Seki Tsuneyoshi, Sakai N

    ICHC2012, Aug. 2012, English, 国際組織細胞化学会, 京都, International conference

    Public symposium

  • Glial functions of Rho-family small GTPases during cerebellar development and after CNS injury

    Ueyama Takehiko, Kondoh T, Kohta M, Kohmura Eiji, Hayashibe M, Nakamura T, Sakahara M, Kassai H, Nakao K, Aiba A, Saito Naoaki

    ICHC2012, Aug. 2012, English, 国際組織細胞化学会, 京都, International conference

    Poster presentation

  • Brain damage and Rho Family GTPases

    Ueyama Takehiko, Saito Naoaki

    第85回日本薬理学会年会, Mar. 2012, English, 日本薬理学会, 京都, Domestic conference

    Public symposium

  • 高輝度放射光を用いたノックアウトマウスの微小脳損傷モデル:小脳顆粒細胞の脱落に低分子量Gタンパク質Racが関与する

    Kota Masaaki, 近藤 威, 梅谷 啓二, Ueyama Takehiko, Saito Naoaki, Kohmura Eiji

    第12回日本分子脳神経外科学会, Oct. 2011, Japanese, 日本分子脳神経外科学会, 横浜, Domestic conference

    Oral presentation

  • 高輝度放射光を用いたノックアウトマウスの微小脳損傷モデル:小脳顆粒細胞の脱落に低分子量Gタンパク質Racが関与する

    Kota Masaaki, 近藤 威, 梅谷 啓二, Ueyama Takehiko, Saito Naoaki, Kohmura Eiji

    社団法人日本脳神経外科学会第70回学術総会, Oct. 2011, Japanese, 日本脳神経外科学会, 横浜, Domestic conference

    Poster presentation

  • Histological analysis of small G proteins using region spscific knock-out mice

    Ueyama Takehiko, Saito Naoaki

    第52回組織細胞化学会年会, Sep. 2011, Japanese, 日本組織細胞化学会, 金沢, Domestic conference

    Public symposium

  • マウス初代培養表皮細胞におけるRac3のRac1代償機能

    清水 彩, Ueyama Takehiko, 坂原 瑞穂, 饗場 篤, Saito Naoaki

    第82回日本薬理学会年会, Mar. 2009, Japanese, パシフィコ横浜, Domestic conference

    Oral presentation

  • NADPH oxidaseファミリーの活性化機構とその生理機能

    Ueyama Takehiko

    第3回グローバルCOE研究討論会, Jul. 2008, Japanese, 淡路島, Domestic conference

    Oral presentation

  • RalAはeta PKCによるケラチノサイトの扁平化に必須である

    Shirai Yasuhito, 盛岡 祥子, 大辻 千裕, Yoshino Ken-ichi, 酒井 規雄, Ueyama Takehiko, Saito Naoaki

    第113回日本薬理学会近畿部会, Jun. 2008, Japanese, 岡山, Domestic conference

    Oral presentation

  • Sequential targeting of cytosolic phox proteins to phagosomes through regulated adaptor proteins during Fc gamma R-mediated phagocytosis.

    Ueyama Takehiko, Kusakabe Tomoko, Karasawa Satoshi, Kawasaki Takumi, Shimizu Aya, Son Jeong-hyun, Leto L. Thomas

    第81回日本薬理学会年会, Mar. 2008, Japanese, パシフィコ横浜, Domestic conference

    Oral presentation

  • Differential regulation of dual oxidase 1 by dual activator 1 splicing variants.

    Tsujibe Satoshi, Ueyama Takehiko, Shirai Yasuhito, Leto L. Thomas, Saito Naoaki

    第81回日本薬理学会年会, Mar. 2008, Japanese, パシフィコ横浜, Domestic conference

    Oral presentation

  • Sequential binding of cytosolic phox complex to phagosomes through

    Ueyama Takehiko, Kusakabe Tomoko, Karasawa Satoshi, Kawasaki Takumi, Shimizu Aya, Son Jeong-hyun, Leto L.Thomas, Miyawaki Atsushi, Saito Naoaki

    Gordon Research Conference, 2008, English, ボストン, International conference

    Poster presentation

  • 脳梗塞における細胞模型エストロゲン受容体を介したPKCの神経保護作用

    Ueyama Takehiko, Kondo Takeshi, Kohmura Eiji

    第66回社団法人日本脳神経外科学会総会, Oct. 2007, Japanese, 社団法人日本脳神経外科学会, 東京, Domestic conference

    Oral presentation

  • Clarification of regulatory mechanism of Orail(SOCs)by PKC.

    Kawasaki Takumi, Ueyama Takehiko, Shirai Yasuhito

    14th Congress of Ca2+ binding proteins & Ca2+ function in health & disease, Oct. 2007, English, La Palma Spain, International conference

    Poster presentation

  • A regulated adaptor function of p40phox:Intramolecularinteraction(PX-PB1 domain)within p40phox.

    Ueyama Takehiko, Tatsuno Toshihiko, Kawasaki Takumi, Leto L. Thomas, Saito Naoaki

    Gordon Research Conference, Jun. 2007, English, ロードアイランド, International conference

    Poster presentation

  • 新規蛍光蛋白システムを用いたphox蛋白内の特異的結合の解明:p40phoxの分子内結合に注目して

    Kusakabe Tomoko, Shimizu Aya, Ueyama Takehiko, Kawasaki Takumi, Shirai Yasuhito, 唐澤 智司, Miyawaki Atsushi, Saito Naoaki

    第80回日本薬理学会年会, Mar. 2007, Japanese, 名古屋国際会議場, Domestic conference

    Oral presentation

  • 新規蛍光蛋白システムを用いたphox蛋白内の特異的結合の解明:p40phoxの分子内結合に注目して

    Kusakabe Tomoko, Shimizu Aya, Ueyama Takehiko, Kawasaki Takumi, Shirai Yasuhito, Karasawa Tomoshi, Saito Naoaki

    第80回日本薬理学会年会, Mar. 2007, Japanese, 名古屋国際会議場, Domestic conference

    Oral presentation

  • Nox2活性化におけるp40phoxのp67phoxに対するキャリア一蛋白としての機能獲得メカニズム:p40phoxのPX-PB1 domainを介した分子内結合の切断

    Ueyama Takehiko, Tatsuno Toshihiko, Kawasaki Takumi, Tsujibe Satoshi, Sumimoto Hideki, Leto L. Thomas, Saito Naoaki

    第80回日本薬理学会年会, Mar. 2007, Japanese, 名古屋国際会議場, Domestic conference

    Oral presentation

  • Nox2活性化におけるp40phoxのp67phoxに対するキャリア一蛋白としての機能獲得メカニズム:p40phoxのPX-PB1 domainを介した分子内結合の切断

    Ueyama Takehiko, Tatsuno Toshihiko, Kawasaki Takumi, Tsujibe Satoshi, Sumomoto Hideki, Thomas L. Leto, Saito Naoaki

    第80回日本薬理学会年会, Mar. 2007, Japanese, 名古屋国際会議場, Domestic conference

    Oral presentation

  • 貪食細胞(Nox2)と内耳上皮細胞(Nox3)おける活性化型NADPH oxidase複合体形成機構---Nox2:p40phoxのアダプターとしての機能獲得メカニズム---

    Ueyama Takehiko, Tatsuno Toshihiko, Kawasaki Takumi, Thomas L. Leto, Saito Naoaki

    第41回食細胞機能異常症研究会, Dec. 2006, Japanese, 東京経団連会館, Domestic conference

    Oral presentation

  • ジアシルグリセロールキナーゼによるクラスリン依存性エンドサイトーシスの制御

    Kawasaki Takumi, Kobayashi Tsuyoshi, 上山 健彦, Shirai Yasuhito, Saito Naoaki

    第110回日本薬理学会近畿部会, Nov. 2006, Japanese, ぱるるプラザ京都, Domestic conference

    Oral presentation

  • 貪食細胞における活性化型NADPH oxidase複合体形成機構の可視化---p40phoxのアダプター蛋白としての機能獲得メカニズムの解明---

    Ueyama Takehiko, Tatsuno Toshihiko, Kawasaki Takunmi, Sumimoto Hideki, Tomas L. Leto, Saito Naoaki

    生理研シンポジウム, Oct. 2006, Japanese, 岡崎カンファレンスセンター, Domestic conference

    Oral presentation

  • Rac1 is an essential component for activation of multi-component Nox1-based NADPH oxidase.

    Ueyama Takehiko, Geiszt M, Saito Naoaki, Leto L.Thomas

    20th IUBMB International Congress of Biochemistry and Molecular Biology and 11th FAOBMB Congress, Jun. 2006, English, 国立京都国際会館, Domestic conference

    Poster presentation

  • NADPHオキシダーゼ複合体の形成メカニズムの解析

    TATSUNO Toshihiko, UYAMA Takehiko, TUJIBE Satoshi, SHIRAI Yasuhitp, SAITO Naoaki

    第79回日本薬理学会年会, Mar. 2006, Japanese, Domestic conference

    Oral presentation

  • ライブイメージングによる貪食作用におけるRacサブタイプの機能解析

    SITO Naoaki, UEYAMA Takehiko, TATSUNO Toshihiko, SUMITOMO Hideki, Michelle LENNARTZ

    第58回日本薬理学会西南部会, Nov. 2005, Japanese, Domestic conference

    Oral presentation

  • 局所脳虚血時の神経保護作用におけるγPKCの関与

    林 成人, UEYAMA, Takehiko, 八木 圭子, 梶本 武利, KOUMURA, Eiji, Saito Naoaki

    第16回日本脳循環代謝学会総会, Sep. 2004, Japanese, 日本脳循環代謝学会, 大宮, Domestic conference

    Oral presentation

Association Memberships

  • 米国免疫学会

    2013 - Present
  • 日本生化学会

    2009 - Present
  • 日本薬理学会

    2001 - Present
  • 北米神経科学会

    2000 - Present
  • 日本脳神経外科学会

    1992 - Present

Research Projects