*A space between the first name and last name, please enter



FacultyDepartment of Life Science / Graduate School of Science and Engineering Research
PositionAssociate Professor
Commentator Guide
Last Updated :2020/07/08

Education and Career

Academic & Professional Experience

  •   2009 ,  - 2013 , Osaka University

Research Activities

Research Areas

  • Life sciences, Cell biology
  • Life sciences, Immunology

Research Interests


Published Papers

  • Endogenous membrane receptors labeling by reactive cytokines/growth factors to chase their dynamics in live cells, Takaoka Y, Uchinomiya S, Kobayashi D, Endo M, Hayashi T, Fukuyama Y, Hayasaka H, Miyasaka M, Ueda T, Shimada I, Hamachi I, Chem, Chem, 4(6), 1451 - 1464, Jun. 2018 , Refereed
  • Regulation of CCR7-dependent cell migration through CCR7 homodimer formation, Daichi Kobayashi, Masataka Endo, Hirotaka Ochi, Hironobu Hojo, Masayuki Miyasaka, Haruko Hayasaka, SCIENTIFIC REPORTS, SCIENTIFIC REPORTS, 7(1), 8536, Aug. 2017 , Refereed
    Summary:The chemokine receptor CCR7 contributes to various physiological and pathological processes including T cell maturation, T cell migration from the blood into secondary lymphoid tissues, and tumor cell metastasis to lymph nodes. Although a previous study suggested that the efficacy of CCR7 ligand-dependent T cell migration correlates with CCR7 homo- and heterodimer formation, the exact extent of contribution of the CCR7 dimerization remains unclear. Here, by inducing or disrupting CCR7 dimers, we demonstrated a direct contribution of CCR7 homodimerization to CCR7-dependent cell migration and signaling. Induction of stable CCR7 homodimerization resulted in enhanced CCR7-dependent cell migration and CCL19 binding, whereas induction of CXCR4/CCR7 heterodimerization did not. In contrast, dissociation of CCR7 homodimerization by a novel CCR7-derived synthetic peptide attenuated CCR7-dependent cell migration, ligand-dependent CCR7 internalization, ligand-induced actin rearrangement, and Akt and Erk signaling in CCR7-expressing cells. Our study indicates that CCR7 homodimerization critically regulates CCR7 ligand- dependent cell migration and intracellular signaling in multiple cell types.
  • [Regulation of immune cell and cancer cell trafficking by multiple chemokines]., Hayasaka H, Okada M, Bai Z, Kuroda Y, Yoshida J, Miyasaka M, Seikagaku. The Journal of Japanese Biochemical Society, Seikagaku. The Journal of Japanese Biochemical Society, 83(10), 930 - 937, Oct. 2011 , Refereed
  • CXC chemokine ligand 12 promotes CCR7-dependent naive T cell trafficking to lymph nodes and Peyer's patches., Bai Z, Hayasaka H, Kobayashi M, Li W, Guo Z, Jang MH, Kondo A, Choi BI, Iwakura Y, Miyasaka M, Journal of immunology (Baltimore, Md. : 1950), Journal of immunology (Baltimore, Md. : 1950), 182(3), 1287 - 1295, Feb. 2009 , Refereed
  • Binding of lymphoid chemokines to collagen IV that accumulates in the basal lamina of high endothelial venules: its implications in lymphocyte trafficking., Yang BG, Tanaka T, Jang MH, Bai Z, Hayasaka H, Miyasaka M, Journal of immunology (Baltimore, Md. : 1950), Journal of immunology (Baltimore, Md. : 1950), 179(7), 4376 - 4382, Oct. 2007 , Refereed
  • Requirements for signal delivery through CD44: analysis using CD44-Fas chimeric proteins., Ishiwatari-Hayasaka H, Fujimoto T, Osawa T, Hirama T, Toyama-Sorimachi N, Miyasaka M, Journal of immunology (Baltimore, Md. : 1950), Journal of immunology (Baltimore, Md. : 1950), 163(3), 1258 - 1264, Aug. 1999 , Refereed
  • Lysophosphatidic acid receptors LPA(4) and LPA(6) differentially promote lymphocyte transmigration across high endothelial venules in lymph nodes, Erina Hata, Naoko Sasaki, Akira Takeda, Kazuo Tohya, Eiji Umemoto, Noriyuki Akahoshi, Satoshi Ishii, Kana Bando, Takaya Abe, Kuniyuki Kano, Junken Aoki, Haruko Hayasaka, Masayuki Miyasaka, INTERNATIONAL IMMUNOLOGY, INTERNATIONAL IMMUNOLOGY, 28(6), 283 - 292, Jun. 2016 , Refereed
    Summary:Naive lymphocytes continuously migrate from the blood into lymph nodes (LNs) via high endothelial venules (HEVs). To extravasate from the HEVs, lymphocytes undergo multiple adhesion steps, including tethering, rolling, firm adhesion and transmigration. We previously showed that autotaxin (ATX), an enzyme that generates lysophosphatidic acid (LPA), is highly expressed in HEVs, and that the ATX/LPA axis plays an important role in the lymphocyte transmigration across HEVs. However, the detailed mechanism underlying this axis's involvement in lymphocyte transmigration has remained ill-defined. Here, we show that two LPA receptors, LPA(4) and LPA(6), are selectively expressed on HEV endothelial cells (ECs) and that LPA(4) plays a major role in the lymphocyte transmigration across HEVs in mice. In the absence of LPA(4) expression, lymphocytes accumulated heavily within the HEV EC layer, compared to wild-type (WT) mice. This accumulation was also observed in the absence of LPA(6) expression, but it was less pronounced. Adoptive transfer experiments using WT lymphocytes revealed that the LPA(4) deficiency in ECs specifically compromised the lymphocyte transmigration process, whereas the effect of LPA(6) deficiency was not significant. These results indicate that the signals evoked in HEV ECs via the LPA(4) and LPA(6) differentially regulate lymphocyte extravasation from HEVs in the peripheral LNs.
  • Fibroblastic reticular cell-derived lysophosphatidic acid regulates confined intranodal T-cell motility, Akira Takeda, Daichi Kobayashi, Keita Aoi, Naoko Sasaki, Yuki Sigiura, Hidemitsu Igarashi, Kazuo Tohya, Asuka Inoue, Erina Hata, Noriyuki Akahoshi, Haruko Hayasaka, Junichi Kikuta, Elke Scandella, Brukhard Ludewig, Satoshi Ishii, Junken Aoki, Makoto Suematsu, Masaru Ishii, Kiyoshi Takeda, Sirpa Jalkanen, Masayuki Miyasaka, Eiji Umemoto, ELIFE, ELIFE, 5, e10561, Feb. 2016 , Refereed
    Summary:Lymph nodes (LNs) are highly confined environments with a cell-dense three-dimensional meshwork, in which lymphocyte migration is regulated by intracellular contractile proteins. However, the molecular cues directing intranodal cell migration remain poorly characterized. Here we demonstrate that lysophosphatidic acid (LPA) produced by LN fibroblastic reticular cells (FRCs) acts locally to LPA(2) to induce T-cell motility. In vivo, either specific ablation of LPA-producing ectoenzyme autotaxin in FRCs or LPA(2) deficiency in T cells markedly decreased intranodal T cell motility, and FRC-derived LPA critically affected the LPA(2)-dependent T-cell motility. In vitro, LPA activated the small GTPase RhoA in T cells and limited T-cell adhesion to the underlying substrate via LPA(2). The LPA-LPA(2) axis also enhanced T-cell migration through narrow pores in a three-dimensional environment, in a ROCK-myosin II-dependent manner. These results strongly suggest that FRC-derived LPA serves as a cell-extrinsic factor that optimizes T-cell movement through the densely packed LN reticular network.
  • Divergence of Vascular Specification in Visceral Lymphoid OrgansGenetic Determinants and Differentiation Checkpoints, Zoltan Kellermayer, Haruko Hayasaka, Bela Kajtar, Diana Simon, Eloy F. Robles, Jose A. Martinez-Climent, Peter Balogh, INTERNATIONAL REVIEWS OF IMMUNOLOGY, INTERNATIONAL REVIEWS OF IMMUNOLOGY, 35(6), 489 - 502, 2016 , Refereed
    Summary:Despite their functional similarities, peripheral lymphoid tissues are remarkably different according to their developmental properties and structural characteristics, including their specified vasculature. Access of leukocytes to these organs critically depends on their interactions with the local endothelium, where endothelial cells are patterned to display a restricted set of adhesion molecules and other regulatory compounds necessary for extravasation. Recent advances in high throughput analyses of highly purified endothelial subsets in various lymphoid tissues as well as the expansion of various transgenic animal models have shed new light on the transcriptional complexities of lymphoid tissue vascular endothelium. This review is aimed at providing a comprehensive analysis linking the functional competence of spleen and intestinal lymphoid tissues with the developmental programming and functional divergence of their vascular specification, with particular emphasis on the transcriptional control of endothelial cells exerted by Nkx2.3 homeodomain transcription factor.
  • The HIV-1 Gp120/CXCR4 Axis Promotes CCR7 Ligand-Dependent CD4 T Cell Migration: CCR7 Homo- and CCR7/CXCR4 Hetero-Oligomer Formation as a Possible Mechanism for Up-Regulation of Functional CCR7, Haruko Hayasaka, Daichi Kobayashi, Hiromi Yoshimura, Emi E. Nakayama, Tatsuo Shioda, Masayuki Miyasaka, PLOS ONE, PLOS ONE, 10(2), e0117454, Feb. 2015 , Refereed
    Summary:During human immunodeficiency virus (HIV) infection, enhanced migration of infected cells to lymph nodes leads to efficient propagation of HIV-1. The selective chemokine receptors, including CXCR4 and CCR7, may play a role in this process, yet the viral factors regulating chemokine-dependent T cell migration remain relatively unclear. The functional cooperation between the CXCR4 ligand chemokine CXCL12 and the CCR7 ligand chemokines CCL19 and CCL21 enhances CCR7-dependent T cell motility in vitro as well as cell trafficking into the lymph nodes in vivo. In this study, we report that a recombinant form of a viral CXCR4 ligand, X4-tropic HIV-1 gp120, enhanced the CD4 T cell response to CCR7 ligands in a manner dependent on CXCR4 and CD4, and that this effect was recapitulated by HIV-1 virions. HIV-1 gp120 significantly enhanced CCR7-dependent CD4 T cell migration from the footpad of mice to the draining lymph nodes in in vivo transfer experiments. We also demonstrated that CXCR4 expression is required for stable CCR7 expression on the CD4 T cell surface, whereas CXCR4 signaling facilitated CCR7 ligand binding to the cell surface and increased the level of CCR7 homo-as well as CXCR4/CCR7 hetero-oligomers without affecting CCR7 expression levels. Our findings indicate that HIV-evoked CXCR4 signaling promotes CCR7-dependent CD4 T cell migration by up-regulating CCR7 function, which is likely to be induced by increased formation of CCR7 homo-and CXCR4/CCR7 hetero-oligomers on the surface of CD4 T cells.
  • Dynamic Changes in Endothelial Cell Adhesion Molecule Nepmucin/CD300LG Expression under Physiological and Pathological Conditions, Eiji Umemoto, Akira Takeda, Soojung Jin, Zhijuan Luo, Naoki Nakahogi, Haruko Hayasaka, Chun Man Lee, Toshiyuki Tanaka, Masayuki Miyasaka, PLOS ONE, PLOS ONE, 8(12), e83681, Dec. 2013 , Refereed
    Summary:Vascular endothelial cells often change their phenotype to adapt to their local microenvironment. Here we report that the vascular endothelial adhesion molecule nepmucin/CD300LG, which is implicated in lymphocyte binding and transmigration, shows unique expression patterns in the microvascular endothelial cells of different tissues. Under physiological conditions, nepmucin/CD300LG was constitutively and selectively expressed at the luminal surface of the small arterioles, venules, and capillaries of most tissues, but it was only weakly expressed in the microvessels of the splenic red pulp and thymic medulla. Furthermore, it was barely detectable in immunologically privileged sites such as the brain, testis, and uterus. The nepmucin/CD300LG expression rapidly decreased in lymph nodes receiving acute inflammatory signals, and this loss was mediated at least in part by TNF-alpha. It was also down-regulated in tumors and tumor-draining lymph nodes, indicating that nepmucin/CD300LG expression is negatively regulated by locally produced signals under these circumstances. In contrast, nepmucin/CD300LG was induced in the high endothelial venule-like blood vessels of chronically inflamed pancreatic islets in an animal model of non-obese diabetes. Interestingly, the activated CD4(+) T cells infiltrating the inflamed pancreas expressed high levels of the nepmucin/CD300LG ligand(s), supporting the idea that nepmucin/CD300LG and its ligand interactions are locally involved in pathological T cell trafficking. Taken together, these observations indicate that the nepmucin/CD300LG expression in microvascular endothelial cells is influenced by factor(s) that are locally produced in tissues, and that its expression is closely correlated with the level of leukocyte infiltration in certain tissues.
  • Constitutive Lymphocyte Transmigration across the Basal Lamina of High Endothelial Venules Is Regulated by the Autotaxin/Lysophosphatidic Acid Axis, Zhongbin Bai, Linjun Cai, Eiji Umemoto, Akira Takeda, Kazuo Tohya, Yutaka Komai, Punniyakoti Thanikachalam Veeraveedu, Erina Hata, Yuki Sugiura, Akiko Kubo, Makoto Suematsu, Haruko Hayasaka, Shinichi Okudaira, Junken Aoki, Toshiyuki Tanaka, Harald M. H. G. Albers, Huib Ovaa, Masayuki Miyasaka, JOURNAL OF IMMUNOLOGY, JOURNAL OF IMMUNOLOGY, 190(5), 2036 - 2048, Mar. 2013 , Refereed
    Summary:Lymphocyte extravasation from the high endothelial venules (HEVs) of lymph nodes is crucial for the maintenance of immune homeostasis, but its molecular mechanism remains largely unknown. In this article, we report that lymphocyte transmigration across the basal lamina of the HEVs is regulated, at least in part, by autotaxin (ATX) and its end-product, lysophosphatidic acid (LPA). ATX is an HEV-associated ectoenzyme that produces LPA from lysophosphatidylcholine (LPC), which is abundant in the systemic circulation. In agreement with selective expression of ATX in HEVs, LPA was constitutively and specifically detected on HEVs. In vivo, inhibition of ATX impaired the lymphocyte extravasation from HEVs, inducing lymphocyte accumulation within the endothelial cells (ECs) and sub-EC compartment; this impairment was abrogated by LPA. In vitro, both LPA and LPC induced a marked increase in the motility of HEV ECs; LPC's effect was abrogated by ATX inhibition, whereas LPA's effect was abrogated by ATX/LPA receptor inhibition. In an in vitro transmigration assay, ATX inhibition impaired the release of lymphocytes that had migrated underneath HEV ECs, and these defects were abrogated by LPA. This effect of LPA was dependent on myosin II activity in the HEV ECs. Collectively, these results strongly suggest that HEV-associated ATX generates LPA locally; LPA, in turn, acts on HEV ECs to increase their motility, promoting dynamic lymphocyte-HEV interactions and subsequent lymphocyte transmigration across the basal lamina of HEVs at steady state. The Journal of Immunology, 2013, 190: 2036-2048.
  • Constitutive Plasmacytoid Dendritic Cell Migration to the Splenic White Pulp Is Cooperatively Regulated by CCR7-and CXCR4-Mediated Signaling, Eiji Umemoto, Kazuhiro Otani, Takashi Ikeno, Noel Verjan Garcia, Haruko Hayasaka, Zhongbin Bai, Myoung Ho Jang, Toshiyuki Tanaka, Takashi Nagasawa, Koichi Ueda, Masayuki Miyasaka, JOURNAL OF IMMUNOLOGY, JOURNAL OF IMMUNOLOGY, 189(1), 191 - 199, Jul. 2012 , Refereed
    Summary:Although the spleen plays an important role in host defense against infection, the mechanism underlying the migration of the innate immune cells, plasmacytoid dendritic cells (pDCs), into the spleen remains ill defined. In this article, we report that pDCs constitutively migrate into the splenic white pulp (WP) in a manner dependent on the chemokine receptors CCR7 and CXCR4. In CCR7-deficient mice and CCR7 ligand-deficient mice, compared with wild-type (WT) mice, substantially fewer pDCs were found in the periarteriolar lymphoid sheath of the splenicWPunder steady-state conditions. In addition, the migration of adoptively transferred CCR7-deficient pDCs into the WP was significantly worse than that of WT pDCs, supporting the idea that pDC trafficking to the splenic WP requires CCR7 signaling. WT pDCs responded to a CCR7 ligand with modest chemotaxis and ICAM-1 binding in vitro, and priming with the CCR7 ligand enabled the pDCs to migrate efficiently toward low concentrations of CXCL12 in a CXCR4-dependent manner, raising the possibility that CCR7 signaling enhances CXCR4-mediated pDC migration. In agreement with this hypothesis, CCL21 and CXCL12 were colocalized on fibroblastic reticular cells in the T cell zone and in the marginal zone bridging channels, through which pDCs appeared to enter the WP. Furthermore, functional blockage of CCR7 and CXCR4 abrogated pDC trafficking into the WP. Collectively, these results strongly suggest that pDCs employ both CCR7 and CXCR4 as critical chemokine receptors to migrate into the WP under steady-state conditions. The Journal of Immunology, 2012, 189: 191-199.
  • Novel Regulators of Lymphocyte Trafficking across High Endothelial Venules, Eiji Umemoto, Haruko Hayasaka, Zhongbin Bai, Linjun Cai, Sari Yonekura, Xiaodong Peng, Akira Takeda, Kazuo Tohya, Masayuki Miyasaka, CRITICAL REVIEWS IN IMMUNOLOGY, CRITICAL REVIEWS IN IMMUNOLOGY, 31(2), 147 - 169, 2011 , Refereed
    Summary:The physiological recruitment of circulating lymphocytes from the blood into secondary lymphoid tissues is an essential homeostatic mechanism for the immune system because it allows lymphocytes to encounter efficiently both their specific cognate antigen and the regulatory cells with which they need to interact, to initiate, maintain, and terminate immune responses appropriately. This constitutive lymphocyte trafficking is mediated by high endothelial venules (HEVs), which are present in secondary lymphoid tissues other than the spleen. There is growing evidence that lymphocyte trafficking across HEVs involves at least three steps, namely, (i) tethering/rolling, (ii) arrest/firm adhesion/intraluminal crawling, and (iii) transendothelial migration (TEM). Although the mechanisms underlying the first two steps have been determined relatively well, the mechanism regulating TEM is only partially understood. In particular, the molecular mechanism driving lymphocyte movement from the apical to the basolateral surface of the endothelial cells (ECs) of HEVs remains ill defined. This step is crucial for successful lymphocyte extravasation, and is thus an important target for therapeutic intervention in various immunological diseases. Here, we review the molecular mechanisms governing lymphocyte-HEV interactions, and highlight possible roles for two HEV proteins, i.e., nepmucin/CD300g and autotaxin, in lymphocyte TEM.
  • FRNK Expression Promotes Smooth Muscle Cell Maturation During Vascular Development and After Vascular Injury, Rebecca L. Sayers, Liisa J. Sundberg-Smith, Mauricio Rojas, Haruko Hayasaka, J. Thomas Parsons, Christopher P. Mack, Joan M. Taylor, ARTERIOSCLEROSIS THROMBOSIS AND VASCULAR BIOLOGY, ARTERIOSCLEROSIS THROMBOSIS AND VASCULAR BIOLOGY, 28(12), 2115 - U40, Dec. 2008 , Refereed
    Summary:Objective - Smooth muscle cell (SMC) differentiation is a dynamic process that must be tightly regulated for proper vascular development and to control the onset of vascular disease. Our laboratory previously reported that a specific focal adhesion kinase (FAK) inhibitor termed FRNK (FAK Related Non-Kinase) is selectively expressed in large arterioles when SMCs are transitioning from a synthetic to contractile phenotype and that FRNK inhibits FAK-dependent SMC proliferation and migration. Herein, we sought to determine whether FRNK expression modulates SMC phenotypes in vivo. Methods and Results - We present evidence that FRNK -/-mice exhibit attenuated SM marker gene expression during postnatal vessel growth and after vascular injury. We also show that FRNK expression is regulated by transforming growth factor (TGF)-beta and that forced expression of FRNK in cultured cells induces serum- and TGF-beta-stimulated SM marker gene expression, whereas FRNK deletion or expression of a constitutively activated FAK variant attenuated SM gene transcription. Conclusions - These data highlight the possibility that extrinsic signals regulate the SMC gene profile, at least in part, by modulating the expression of FRNK and that tight regulation of FAK activity by FRNK is important for proper SMC differentiation during development and after vascular injury. (Arterioscler Thromb Vasc Biol. 2008;28:2115-2122.)
  • Involvement of the lysophosphatidic acid-generating enzyme autotaxin in lymphocyte-endothelial cell interactions., Tae Nakasaki, Toshiyuki Tanaka, Shinichi Okudaira, Michi Hirosawa, Eiji Umemoto, Kazuhiro Otani, Soojung Jin, Zhongbin Bai, Haruko Hayasaka, Yoshinori Fukui, Katsuyuki Aozasa, Naoya Fujita, Takashi Tsuruo, Keiichi Ozono, Junken Aoki, Masayuki Miyasaka, The American journal of pathology, The American journal of pathology, 173(5), 1566 - 76, Nov. 2008 , Refereed
    Summary:Autotaxin (ATX) is a secreted protein with lysophospholipase D activity that generates lysophosphatidic acid (LPA) from lysophosphatidylcholine. Here we report that functional ATX is selectively expressed in high endothelial venules (HEVs) of both lymph nodes and Peyer's patches. ATX expression was developmentally regulated and coincided with lymphocyte recruitment to the lymph nodes. In adults, ATX expression was independent of HEV-expressed chemokines such as CCL21 and CXCL13, innate immunity signals including those via TLR4 or MyD88, and of the extent of lymphocyte trafficking across the HEVs. ATX expression was induced in venules at sites of chronic inflammation. Receptors for the ATX enzyme product LPA were constitutively expressed in HEV endothelial cells (ECs). In vitro, LPA induced strong morphological changes in HEV ECs. Forced ATX expression caused cultured ECs to respond to lysophosphatidylcholine, up-regulating lymphocyte binding to the ECs in a LPA receptor-dependent manner under both static and flow conditions. Although in vivo depletion of circulating ATX did not affect lymphocyte trafficking into the lymph nodes, we surmise, based on the above data, that ATX expressed by HEVs acts on HEVs in situ to facilitate lymphocyte binding to ECs and that ATX in the general circulation does not play a major role in this process. Tissue-specific inactivation of ATX will verify this hypothesis in future studies of its mechanism of action.
  • Disruption of FRNK expression by gene targeting of the intronic promoter within the focal adhesion kinase gene, Haruko Hayasaka, Karen H. Martin, E. Daniel Hershey, J. Thomas Parsons, JOURNAL OF CELLULAR BIOCHEMISTRY, JOURNAL OF CELLULAR BIOCHEMISTRY, 102(4), 947 - 954, Nov. 2007 , Refereed
    Summary:FRNK, a non-catalytic variant of focal adhesion kinase (FAK), is expressed in major blood vessels throughout mouse development and is postulated to play a role in regulating cell adhesion and signaling in vascular smooth muscle cells (VSMCS). The FRNK transcriptional start site lies within an intron of the FAK gene, suggesting that the FRNK gene is a "gene within a gene". Here, we identified a 1kb intronic sequence of the FAK gene that is necessary for endogenous FRNK expression. Deletion of this sequence in gene-targeted mice abolished FRNK expression, showing the direct involvement of the FAK intron in the regulation of FRNK expression. The level of FAK expression was normal in the FRNK-deficient mice, indicating that FAK and FRNK are transcriptionally regulated by distinct promoters. The FRNK-deficient mice were viable, fertile, and displayed no obvious histological abnormalities in any of the major blood vessels. Western blot analysis showed that FRNK-deficientand wild-type (WT) cells had comparable levelsof steady-state and adhesion -dependent FAK autophosphorylation. Despite the fact that ectopic expression of FRNK suppresses focal adhesion formation in cultured cells, these results suggest that endogenous FRNK is not essential for development or the formation of the mouse vasculature.
  • Ligand-induced structural changes of the CD44 hyaluronan-binding domain revealed by NMR, Mitsuhiro Takeda, Shinji Ogino, Ryo Umemoto, Masayoshi Sakakura, Masahiro Kajiwara, Kazuki N. Sugahara, Haruko Hayasaka, Masayuki Miyasaka, Hiroaki Terasawa, Ichio Shimada, JOURNAL OF BIOLOGICAL CHEMISTRY, JOURNAL OF BIOLOGICAL CHEMISTRY, 281(52), 40089 - 40095, Dec. 2006 , Refereed
    Summary:CD44, a major cell surface receptor for hyaluronan (HA), contains a functional domain responsible for HA binding at its N terminus (residues 21-178). Accumulating evidence indicates that proteolytic cleavage of CD44 in its extracellular region (residues 21-268) leads to enhanced tumor cell migration and invasion. Hence, understanding the mechanisms underlying the CD44 proteolytic cleavage is important for understanding the mechanism of CD44-mediated tumor progression. Here we present the NMR structure of the HA-binding domain of CD44 in its HA-bound state. The structure is composed of the Link module (residues 32-124) and an extended lobe (residues 21-31 and 125-152). Interestingly, a comparison of its unbound and HA-bound structures revealed that rearrangement of the beta-strands in the extended lobe (residues 143-148) and disorder of the structure in the following C-terminal region (residues 153-169) occurred upon HA binding, which is consistent with the results of trypsin proteolysis studies of the CD44 HA-binding domain. The order-to-disorder transition of the C-terminal region by HA binding may be involved in the CD44-mediated cell migration.
  • FRNK, the autonomously expressed C-terminal region of focal adhesion kinase, is uniquely regulated in vascular smooth muscle: Analysis of expression in transgenic mice, H Hayasaka, K Simon, ED Hershey, K Masumoto, JT Parsons, JOURNAL OF CELLULAR BIOCHEMISTRY, JOURNAL OF CELLULAR BIOCHEMISTRY, 95(6), 1248 - 1263, Aug. 2005 , Refereed
    Summary:FRNK, the autonomously expressed carboxyl-terminal region of focal adhesion kinase(FAK), is expressed in tissues that are rich in vascular smooth muscle cells (VSMCs). Here we report the generation of transgenic mice harboring the putative FRNK promoter fused to LacZ and examine the promoter activity in situ via expression of beta-galactosidase. The transgenic mice exhibited expression of beta-galactosidase predominantly in arterial VSMCs in large and small blood vessels of major organs. Upregulation of beta-galactosidase activity was observed in tunica media following carotid injury, indicating that the FRNK promoter is activated in VSMCs in response to injury. Robust expression of beta-galactosidase in blood vessels was also detected in the developing embryo. However, expression was also observed in the midline, the nose and skin epidermis, indicating distinct transcriptional regulation of the FRNK promoter in embryogenesis. To analyze FRNK expression in vitro, we identified a 116 bp sequence in the FRNK promoter that was sufficient to function as an enhancer when fused to the minimal actin promoter and expressed in cultured smooth muscle cells. Mutation of AP-1 and NF-E2 binding consensus sequences within this element abrogated enhancer activity, supporting the involvement of this promoter element in VSMC expression of FRNK.
  • Chemokines in tumor progression and metastasis, T Tanaka, ZB Bai, Y Srinoulprasert, BG Yang, H Hayasaka, M Miyasaka, CANCER SCIENCE, CANCER SCIENCE, 96(6), 317 - 322, Jun. 2005 , Refereed
    Summary:Although chemokines have been thought of primarily as leukocyte attractants, a growing body of evidence indicates that they also contribute to a number of tumor-related processes, such as tumor cell growth, angiogenesis/angiostasis, local invasion, and metastasis. The current knowledge of the possible involvement of chemokines and their receptors in these cellular events are reviewed here. The operating mechanism of chemokines in relation to metastatic processes in vivo are also discussed.
  • Interaction of neuropeptide Upsilon and Hsp90 through a novel peptide binding region, H Ishiwatari-Hayasaka, M Maruya, AS Sreedhar, TK Nemoto, P Csermely, Yahara, I, BIOCHEMISTRY, BIOCHEMISTRY, 42(44), 12972 - 12980, Nov. 2003 , Refereed
    Summary:Hsp90 is a molecular chaperone that binds and assists refolding of non-native and/or labile polypeptides and also bind various peptides. However, the rules of how Hsp90 recognizes substrates have not been well characterized. By surface plasmon resonance measurements, a physiologically active peptide, neuropeptide Y (NPY), with a strong binding property to Hsp90 was identified from screening of 38 randomly selected peptide candidates. We showed that the carboxy-terminal fragment of NPY (NPY13-36), which forms an amphipathic alpha-helix structure, preserved the strong binding to Hsp90. Immunoprecipitation and immunoblotting using HeLa cell extracts revealed that newly synthesized NPY precursors bound to Hsp90, suggesting that the in vitro binding experiments identified an interactive peptide in vivo. Proteolytic cleavage of the NPY13-36/Hsp90 complex, as well as binding site analysis using deletion mutants of Hsp90, revealed the NPY binding locus on Hsp90alpha as the 192 amino acid region following the N-terminal domain. By electron microscopic analysis using an anti-Hsp90 antibody against the sequence proximal to the highly charged region, we showed that the Hsp90 dimer bound to NPY13-36 at both ends. Mutation of arginine residues in NPY13-36 to alanine abrogated binding to Hsp90. Our studies indicate that the hinge region after the N-terminal domain of Hsp90 and the positive charges on NPY are important for this interaction.
  • Interactions of Hsp90 with histones and related peptides, T Schnaider, J Oikarinen, H Ishiwatari-Hayasaka, Yahara, I, P Csermely, LIFE SCIENCES, LIFE SCIENCES, 65(22), 2417 - 2426, Oct. 1999 , Refereed
    Summary:The 90 kDa heat shock protein (Hsp90) induces the condensation of the chromatin structure [Csermely, P., Kajtar, J., Hollosi, M., Oikarinen, J., and Somogyi, J. (1994) Biochem. Biophys. Res. Commun. 202, 1657-1663]. In our present studies we used surface plasmon resonance measurements to demonstrate that Hsp90 binds histones H1, H2A, H2B, H3 and H4 with high affinity having dissociation constants in the submicromolar range. Strong binding of the C-terminal peptide of histone H1 containing the SPKK-motif and a pentaeicosa-peptide including the Hsp90 bipartite nuclear localization signal sequence was also observed. However, a lysine/arginine-rich peptide of casein, and the lysine-rich platelet factor 4 did not display a significant interaction with Hsp90. Histones and positively charged peptides modulated the Hsp90-associated kinase activity. Interactions between Hsp90, histones, and high mobility group (HMG) protein-derived peptides raise the possibility of the involvement of Hsp90 in chromatin reorganization during steroid action, mitosis, or after cellular stress.
  • Suppression of tumor growth by the 3' untranslated region of mel-18 in 3Y1 cells transformed by the E6 and E7 genes of human papillomavirus type 18, H Ishiwatari, K Nakanishi, G Kondoh, N Hayasaka, Q Li, A Yamashita, H Inoue, A Hakura, CANCER LETTERS, CANCER LETTERS, 117(1), 57 - 65, Jul. 1997 , Refereed
    Summary:By introducing a cDNA library derived from rat embryonic fibroblast cells, we isolated several morphologically flat revertants of rat 3Y1 cells transformed by the E6 and E7 genes of human papillomavirus type 18 (HPV18). From one of the revertants, we recovered a 0.2-kb cDNA, N56, that suppresses the tumor growth of the transformed 3Y1 cells irrespective of the expression of the E6 and E7 genes. The nucleotide sequence of the cDNA was shown to be identical to that of the 3' untranslated region of a putative mammalian polycomb group gene, mel-18. (C) 1997 Elsevier Science Ireland Ltd.
  • Induction of cell death by chimeric L-selectin-Fas receptors, H IshiwatariHayasaka, H Kawashima, T Osawa, S Nagata, M Miyasaka, INTERNATIONAL IMMUNOLOGY, INTERNATIONAL IMMUNOLOGY, 9(4), 627 - 635, Apr. 1997 , Refereed
    Summary:In the present study, we have established a system where engagement of an adhesion molecule triggers a death signal into cells, L-selectin, which is a well characterized adhesion receptor involved in the initial adhesion between lymphocyte and endothelium, was fused to the intracellular domain of an apoptosis-inducing molecule, Fas. Ligation of the chimeric receptors with a carbohydrate ligand for L-selectin, fucoidin or a mAb that recognizes the lectin domain of L-selectin, induced apoptosis in receptor-expressing cells. However, ligation with an anti-L-selectin mAb reactive with a non-ligand binding site did not induce apoptosis, indicating that stimulation through the lectin domain of L-selectin in the chimeric receptor leads to signal delivery. Upon activation L-selectin shows a unique proteolytic cleavage at the membrane proximal site on the extracellular (EC) domain, of which the significance is also unclear. We found that truncations in the EC domain which abrogate the proteolytic cleavage of L-selectin did not influence induction of apoptosis, suggesting that the cleavage on the EC domain itself is not important for the signaling function of the chimeric receptor. This is the first demonstration that an adhesion signal can be converted to a signal that leads to apoptotic cell death.
    Summary:Certain types of human papillomavirus (HPV), such as types 16 and 18, are thought to be responsible for the development of cervical carcinomas. The E6 and E7 genes of these viruses have transforming activities in various cultured cells and their mRNAs and proteins are expressed in almost all cervical carcinoma cells. Inactivation of the tumor suppressor p53 protein by the E6 gene is believed to be critical for transformation by these oncogenic HPVs. To determine whether degradation of the p53 protein is, in fact, sufficient for cellular transformation by the E6 gene, the E6 gene of HPV16 was introduced into human embryonic fibroblasts (HEF) using recombinant murine retrovirus and examined whether reduction of the p53 protein could substitute for the E6 function. It was found that HEF cells transfected with the E6 gene showed an increased saturation density and degraded the p53 protein. However, when expression of the p53 protein in normal HEF cells was suppressed by the antisense oligonucleotide of the p53 gene, growth stimulation was not observed. These results show that the E6 gene stimulates growth of HEF cells, but that this activity involves some other E6 gene-mediated functions than degradation of the p53 protein. (C) 1994 Wiley-Liss, Inc.
    Summary:For determination of the correlation between tumorigenicity and the expression levels or splicing patterns of E6 mRNAs of the human papillomavirus type 16 in established cells, a vector containing the intact E6 open reading frame which expresses both unspliced and spliced mRNAs, one expressing only unspliced E6 mRNA, and one expressing both unspliced and spliced mRNAs but producing only truncated E6 proteins were constructed. In transformation assays and analyses of E6 mRNAs, a higher expression level of unspliced E6 mRNA was found to be closely associated with tumorigenicity. Furthermore, it was also related with anchorage-independent growth and a decreased serum requirement of the cells.
    Summary:A rat embryo fibroblast (REF) cDNA expression library was transfected into 3Y1 cells transformed by human papillomavirus type 18 E6 and E7 genes and 10 flat revertants were isolated. These revertants expressed the same levels of E6 and E7 mRNA as the parent cells, but had greatly reduced ability to form colonies in soft agar. Suppression of transformation was dominant in cell hybrids generated by fusing each revertant with the parental transformed cells. Furthermore, loss of transfected cDNA was observed in re-transformed cell hybrids derived from one flat revertant. Overexpression of the cDNA suppresses the colony-forming efficiency of the cells transformed by E6 and E7 genes.
    Summary:It has been suggested that the two acidic regions around residue 70 and residue 170 in yeast cytochrome c(1) a subunit of ubiquinol-cytochrome c reductase (complex III), interact with cytochrome c in the electron transfer reaction and that the QCR6 protein, the acidic subunit of yeast complex III, enhances this interaction. In order to determine the roles of the acidic regions of cytochrome c(1) more precisely, we introduced several mutations in the two acidic regions and examined their effects on the ability of modified cytochrome c(1) to complement the respiration deficiency of yeast cells lacking only cytochrome c(1) or both cytochrome c(1) and the QCR6 protein. The mutant cytochrome c(1) with the deletion of the first acidic region (Delta 68-80) was still functional in the cytochrome c(1)-deficient strain. Mutant cytochrome c(1) with the deletion of the second acidic region (Delta 168-179) caused a decrease in the complementing ability, but this is probably due to failure in its proteolytic maturation and/or correct assembly into complex III. Mutant cytochrome c(1) with altered charge distribution in the acidic regions (Asp(170)Asp(171)-->Asn(170)Asn(171) or Asp(170)Asp(171)-->Asn(170)Lys(171)) made the cytochrome c(1)-deficient cells respiration-competent. On the other hand, mutant cytochrome c(1) with the deletion of the first acidic region (Delta 68-80) or altered charge distribution in the second region (Asp(170)Asp(171)-->Asn(170)Lys(171)) did not restore the respiration deficiency of the cells lacking not only cytochrome c(1) but also the QCR6 protein. These results indicate that the acidic regions in cytochrome c(1) are essential for the ubiquinol-cytochrome c reductase activity in yeast cells in the absence of the QCR6 protein, and suggest the acidic regions of cytochrome c(1) may promote binding of complex III to cytochrome c in cooperation with the QCR6 protein.

Conference Activities & Talks

  • Functional analysis of the subcapsular sinus floor that allows leukocyte migration from the sinus to the lymph node parenchyma, SASAKI NAOKO, HATA ERINA, UMEMOTO EIJI, HAYASAKA HARUKO, MIYASAKA MASAYUKI, 日本免疫学会総会・学術集会記録,   2014 11 18
  • The role of LPA4/6 receptors in lymphocyte trafficking across high endothelial venules of lymph nodes, HATA ERINA, SASAKI NAOKO, TAKEDA AKIRA, UMEMOTO EIJI, HAYASAKA HARUKO, MIYASAKA MASAYUKI, 日本免疫学会総会・学術集会記録,   2014 11 18
  • The role of LPA receptors on endothelial cells in lymphocyte trafficking across high endothelial venules of lymph nodes, HATA ERINA, SASAKI NAOKO, TAKEDA AKIRA, UMEMOTO EIJI, HAYASAKA HARUKO, MIYASAKA MASAYUKI, 日本免疫学会総会・学術集会記録,   2013 11 18
  • CXCR4 igands promote CCR7-dependent CD4 T cell migration-possible involvement of CCR7 oligomerization, HAYASAKA HARUKO, KOBAYASHI DAICHI, MIYASAKA MASAYUKI, 日本免疫学会総会・学術集会記録,   2013 11 18
  • Migration behavior of naive T cells and activated T cells via afferent lymphatics, HATA ERINA, UMEMOTO EIJI, HAYASAKA HARUKO, MIYASAKA MASAYUKI, 日本免疫学会総会・学術集会記録,   2012 11 12
  • In vivo inhibition of the autotaxin/LPA axis results in inhibition of lymphocyte transmigration across high endothelial venules of lymph nodes, HATA ERINA, BAI ZHONGBIN, CAI LINJUN, UMEMOTO EIJI, HAYASAKA HARUKO, MIYASAKA MASAYUKI, 日本免疫学会総会・学術集会記録,   2011 11 07


  • Review: Novel regulators of lymphocyte trafficking across high endothelial venules, Crit. Rev. Immunol., 31:147-169,   2011
  • Neogenesis and development of the high endothelial venules that mediate lymphocyte trafficking, Haruko Hayasaka, Kanako Taniguchi, Shoko Fukai, Masayuki Miyasaka, CANCER SCIENCE, 101, 11, 2302, 2308,   2010 11 , 10.1111/j.1349-7006.2010.01687.x
    Summary:Physiological recruitment of lymphocytes from the blood into lymph nodes and Peyer's patches is mediated by high endothelial venules (HEV), specialized blood vessels found in secondary lymphoid tissues except for the spleen. The HEV are distinguished from other types of blood vessels by their tall and plump endothelial cells, and by their expression of specific chemokines and adhesion molecules, which all contribute to the selective lymphocyte trafficking across these blood vessels. The development of HEV is ontogenically regulated, and they appear perinatally in the mouse. High endothelial venules can appear ectopically, for instance in chronically inflamed tissues. Given that HEV enable the efficient trafficking of lymphocytes into tissues, the induction of HEV at a tumor site could potentiate tumor-specific immune responses, and the artificial manipulation of HEV neogenesis might thus provide a new tool for cancer immunotherapy. However, the process of HEV development and the mechanisms by which the unique features of HEV are maintained are incompletely understood. In this review, we discuss the process of HEV neogenesis and development during ontogeny, and their molecular requirements for maintaining their unique characteristics under physiological conditions. (Cancer Sci 2010; 101: 2302-2308).
  • Two-State Conformations in the Hyaluronan-Binding Domain Regulate CD44 Adhesiveness under Flow Condition, Shinji Ogino, Noritaka Nishida, Ryo Umemoto, Miho Suzuki, Mitsuhiro Takeda, Hiroaki Terasawa, Joji Kitayama, Masanori Matsumoto, Haruko Hayasaka, Masayuki Miyasaka, Ichio Shimada, STRUCTURE, 18, 5, 649, 656,   2010 05 , 10.1016/j.str.2010.02.010
    Summary:The hyaluronan (HA) receptor CD44 mediates cell adhesion in leukocyte trafficking and tumor metastasis. Our previous nuclear magnetic resonance (NMR) studies revealed that the CD44 hyaluronan-binding domain (HABD) alters its conformation upon HA binding, from the ordered (O) to the partially disordered (PD) conformation. Here, we demonstrate that the HABD undergoes an equilibrium between the O and PD conformations, in either the presence or absence of HA, which explains the seemingly contradictory X-ray and NMR structures of the HA-bound HABD. An HABD mutant that exclusively adopts the PD conformation displayed a higher HA affinity than the wild-type. Rolling of the cells expressing the mutant CD44 was less efficient than those expressing the wild-type, due to the decreased tether frequency and the slow cellular off rate. Considering that the mutant CD44, devoid of the low-affinity state, exhibited impaired rolling, we conclude that the coexistence of the high- and low-affinity states of the HABD is essential for the CD44-mediated rolling.
  • CXCL12 promotes CCR7-dependent naïve T-cell trafficking to lymph nodes and Peyer’s patches, J. Immunol., 182:1287-1295,   2009
  • Heterogeneity of in vitro lymphocyte behaviours in response to different chemokines, Med. J. Osaka Univ., 51:11-20,   2008
  • Plasmacytoid dendritic cells employ multiple cell adhesion molecules sequentially to interact with high endothelial venule cells - molecular basis of their trafficking to lymph nodes, Takahiro Matsutani, Toshiyuki Tanaka, Kazuo Tohya, Kazuhiro Otani, Myoung Ho Jang, Eiji Umemoto, Kanako Taniguchi, Haruko Hayasaka, Koichi Ueda, Masayuki Miyasaka, INTERNATIONAL IMMUNOLOGY, 19, 9, 1031, 1037,   2007 09 , 10.1093/intimm/dxm088
    Summary:Plasmacytoid dendritic cells (pDCs) are natural type I IFN-producing cells found in lymphoid tissues, where they support both innate and adaptive immune responses. They emigrate from the blood to lymph nodes, apparently through high endothelial venules (HEVs), but little is known about the mechanism. We have investigated the molecular mechanisms of pDC migration using freshly isolated DCs and HEV cells. We found that pDCs bound avidly to HEV cells and then transmigrated underneath them. Two observations suggested that these binding and migration steps are differentially regulated. First, treatment of pDCs with pertussis toxin blocked transmigration but not binding. Second, pDCs were able to bind but not to transmigrate under non-HEV endothelial cells, although the binding was observed to both HEV and non-HEV endothelial cells. Antibody inhibition studies indicated that the binding process was mediated by alpha L and alpha 4 integrins on pDCs and by intercellular adhesion molecule (ICAM)-1, ICAM-2 and vascular cell adhesion molecule-1 on HEVs. The transmigration process was also mediated by aL and a4 integrins on pDCs, with junctional adhesion molecule-A on HEV cells apparently serving as an additional ligand for aL integrin. These data show for the first time that pDCs employ multiple adhesion molecules sequentially in the processes of adhesion to and transmigration through HEVs.
  • Binding of lymphoid chemokines to collagen IV that accumulates in the basal lamina of high endothelial venules; its implication in lymphocyte trafficking, J. Immunol., 179:4376-4382, 179:4376-4382, 2007.,   2007
  • Tumor cells enhance their own CD44 cleavage and motility by generating hyaluronan fragments, KN Sugahara, T Hirata, H Hayasaka, R Stern, T Murai, M Miyasaka, JOURNAL OF BIOLOGICAL CHEMISTRY, 281, 9, 5861, 5868,   2006 03 , 10.1074/jbc.M506740200
    Summary:Hyaluronan (HA) is an extracellular matrix glycosaminoglycan that interacts with cell-surface receptors, including CD44. Although HA usually exists as a high molecular mass polymer, HA of a much lower molecular mass that shows a variety of biological activities can be detected under certain pathological conditions, particularly in tumors. We previously reported that low molecular weight HAs (LMW-HAs) of a certain size range induce the proteolytic cleavage of CD44 from the surface of tumor cells and promote tumor cell migration in a CD44-dependent manner. Here, we show that MIA PaCa-2, a human pancreatic carcinoma cell line, secreted hyaluronidases abundantly and generated readily detectable levels of LMW-HAs ranging from similar to 10- to 40-mers. This occurred in the absence of any exogenous stimulation. The tumor-derived HA oligosaccharides were able to enhance CD44 cleavage and tumor cell motility. Inhibition of the CD44-HA interaction resulted in the complete abrogation of these cellular events. These results are consistent with the concept that tumor cells generate HA oligosaccharides that bind to tumor cell CD44 through the expression of their own constitutive hyaluronidases. This enhances their own CD44 cleavage and cell motility, which would subsequently promote tumor progression. Such an autocrine/paracrine-like process may represent a novel activation mechanism that would facilitate and promote the malignant potential of tumor cells.
  • Chemokines in tumor progression and metastasis, T Tanaka, ZB Bai, Y Srinoulprasert, BG Yang, H Hayasaka, M Miyasaka, CANCER SCIENCE, 96, 6, 317, 322,   2005 06 , 10.1111/j.1349-7006.2005.00059.x
    Summary:Although chemokines have been thought of primarily as leukocyte attractants, a growing body of evidence indicates that they also contribute to a number of tumor-related processes, such as tumor cell growth, angiogenesis/angiostasis, local invasion, and metastasis. The current knowledge of the possible involvement of chemokines and their receptors in these cellular events are reviewed here. The operating mechanism of chemokines in relation to metastatic processes in vivo are also discussed.
  • CXCL13 is an arrest chemokine for B cells in high endothelial venules., Blood, 106:2163-2168,   2005

Research Grants & Projects

  • Ministry of Education, Culture, Sports, Science and Technology, Grants-in-Aid for Scientific Research(新学術領域研究(研究領域提案型)), Integrative understanding of biological processes mediated by transient macromolecul ar complexes; New technology for visualizing physiologically metastable states, The role of the management team is to promote and coordinate each project of the Grant-in-Aid for Scientific Research on Innovative Area "Katoteki-Fukugoutai" (transient molecular complex). Specifically, we held the all-member meeting once a year, and the management team meeting on a regular basis. Efforts have been made to spend research funds efficiently by organizing the joint purchase of the reagents, and to promote collaboration between the members. We organized a symposium, and hosted workshops in various domestic conferences. We support the tutorial course for young investigators. We issued newsletters on a regular basis, and wrapped up an annual report at the end of the year.
  • Ministry of Education, Culture, Sports, Science and Technology, Grants-in-Aid for Scientific Research(新学術領域研究(研究領域提案型)), Analysis of the conformational transitions and functional modifications in membrane proteins, In various cancer cells, the expression of adhesion molecules and chemokine receptor shows a positive correlation with the metastatic potential, suggesting a possible involvement of those molecules in cancer metastasis. CD44 plays a role in cell adhesion by binding to its ligand hyaluronate. Chemokines are critical regulators of cell migration in the context of effective and appropriate immune responses, inflammation, angiogenesis and tumor progression. In this research project, we found that the transition of CD44 molecular structure by ligand binding is possibly involved in the CD44-mediated tumor progression. We also found that the dynamics of chemokine receptor localization on the plasma membrane are important for their function in cell migration.
  • Ministry of Education, Culture, Sports, Science and Technology, Grants-in-Aid for Scientific Research(基盤研究(C)), Understanding of the mechanism underlying the tissue-specific differentiation of high endothelial venule endothelial cells, The high endothelial venules (HEVs) are blood vessels specifically found in lymph nodes and Peyer ’ s patches. Although HEVs express specific chemokines/adhesion molecules which mediate lymphocyte t rafficking across the high-walled endothelial cells (HEV -ECs), the mechanism regulating HEVs’ development and maintenance of the unique properties remain unclear. We performed microarray and real -time quantitative PCR analyses of HEV -ECs and non-HEV -ECs inneonatal mice mesenteric LNs, and identified five transcription factors which are over fifty times more abundantly expressed in developing HEV -ECs than in non-HEV -ECs. By immunohistochemical analysis, we found that one of them showed a restricted expression pattern in the nucleus of ECs of a substantial proportion of blood vessels oflymph nodes from E17.5 to the date of birth, which corresponds temporally to HEV -EC development. The gene knockout mice of this transcription factor showed reduced expression of several HEV -associated genes, implying the functionalcontribution of this gene to HEV -EC differentiation.