Details of a Researcher - KOUNO Takayuki

Papers - KOUNO Takayuki

Division display >> ／ All the affair displays 1 - 77 of about 77

Atypical Macropinocytosis Contributes to Malignant Progression: A Review of Recent Evidence in Endometrioid Endometrial Cancer Cells

Takayuki Kohno, Takashi Kojima

Cancers 2022.10 [Refereed]

Authorship： Lead author , Corresponding author

DOI

Translocation of LSR from tricellular corners causes macropinocytosis at cell–cell interface as a trigger for breaking out of contact inhibition

Takayuki Kohno, Takumi Konno, Shin Kikuchi, Masuo Kondoh, Takashi Kojima

The FASEB Journal ( Wiley ) 35 ( 9 ) e21742 2021.09 [Refereed] [International journal]

Authorship： Lead author , Corresponding author

Withdrawal from contact inhibition is necessary for epithelial cancer precursor cells to initiate cell growth and motility. Nevertheless, little is understood about the mechanism for the sudden initiation of cell growth under static conditions. We focused on cellular junctions as one region where breaking out of contact inhibition occurs. In well-differentiated endometrial cancer cells, Sawano, the ligand administration for tricellular tight junction protein LSR, which transiently decreased the robust junction property, caused an abrupt increase in cell motility and consequent excessive multilayered cell growth despite being under contact inhibition conditions. We observed that macropinocytosis essentially and temporarily occurred as an antecedent event for the above process at intercellular junctions without disruption of the junction apparatus but not at the apical plasma membrane. Collectively, we concluded that the formation of macropinocytosis, which is derived from tight junction-mediated signaling, was triggered for the initiation of cell growth in static precancerous epithelium.

DOI PubMed

A resourceful alteration of tricellular tight junction proteins: roles in endometrial cancer progression.

Takayuki Kohno

Tight Junctions: Classification, Structure and Functions. ISBN: 978-1-53617-327-7 2020.02 [Refereed]

Authorship： Lead author , Corresponding author

Role of Tricellular Tight Junction Protein Lipolysis-Stimulated Lipoprotein Receptor (LSR) in Cancer Cells.

Takayuki Kohno, Takumi Konno, Takashi Kojima

International journal of molecular sciences 20 ( 14 ) 2019.07 [Refereed] [International journal]

Authorship： Lead author , Corresponding author

Maintaining a robust epithelial barrier requires the accumulation of tight junction proteins, LSR/angulin-1 and tricellulin, at the tricellular contacts. Alterations in the localization of these proteins temporarily cause epithelial barrier dysfunction, which is closely associated with not only physiological differentiation but also cancer progression and metastasis. In normal human endometrial tissues, the endometrial cells undergo repeated proliferation and differentiation under physiological conditions. Recent observations have revealed that the localization and expression of LSR/angulin-1 and tricellulin are altered in a menstrual cycle-dependent manner. Moreover, it has been shown that endometrial cancer progression affects these alterations. This review highlights the differences in the localization and expression of tight junction proteins in normal endometrial cells and endometrial cancers and how they cause functional changes in cells.

DOI PubMed

The bicellular tensile force sorts the localization of LSRs in bicellular and tricellular junctions.

Takayuki Kohno, Shin Kikuchi, Takafumi Ninomiya, Takashi Kojima

Annals of the New York Academy of Sciences ( WILEY ) 1397 ( 1 ) 185 - 194 2017.06 [Refereed] [International journal]

Authorship： Lead author , Corresponding author

Lipolysis-stimulated lipoprotein receptors (LSRs) localize to tricellular tight junctions. Recent studies have shown that changes in the localization and expression profiles of LSRs are associated with malignancy of endometrial carcinomas, although the precise mechanisms by which malignant progression induces changes in the localization of LSRs are still unknown. In this study, we found that changes in cell tension correlated with alterations in the junctional localization of LSRs in endometrial cancer Sawano cells. At high cell densities, myosin phosphatase target subunit 1 (MYPT1) localized to bicellular junctions, whereas activated myosin regulatory light chain 2 (MRLC2) was dislocated from these regions, suggesting that circumferential tensile forces decreased at high cell densities. Under these conditions, LSRs localized to tricellular junctions. In contrast, a phosphorylated form of MRLC2 localized to bicellular regions, while MYPT1 was excluded from these regions, suggesting that tensile forces formed along the circumferential edge at low cell densities. It is noteworthy that, when cells were cultured under these conditions, LSRs localized to bicellular regions. Upon treatment with a myosin inhibitor, LSR localization in bicellular junctions decreased at low cell densities. Overall, our results indicate that the modulation of cellular tension was involved in the translocation of LSRs from bicellular to tricellular tight junctions.

DOI PubMed

Staurosporine Induces Formation of Two Types of Extra-Long Cell Protrusions: Actin-Based Filaments and Microtubule-Based Shafts

Takayuki Kohno, Takafumi Ninomiya, Shin Kikuchi, Takumi Konno, Takashi Kojima

MOLECULAR PHARMACOLOGY ( AMER SOC PHARMACOLOGY EXPERIMENTAL THERAPEUTICS ) 87 ( 5 ) 815 - 824 2015.05 [Refereed]

Authorship： Lead author , Corresponding author

Staurosporine (STS) has been known as a classic protein kinase C inhibitor and is a broad-spectrum inhibitor targeting over 250 protein kinases. In this study, we observed that STS treatment induced drastic morphologic changes, such as elongation of a very large number of nonbranched, actin-based long cell protrusions that reached up to 30 mu m in an hour without caspase activation or PARP cleavage in fibroblasts and epithelial cells. These cell protrusions were elongated not only from the free cell edge but also from the cell-cell junctions. The elongation of STS-dependent protrusions was required for ATP hydrolysis and was dependent on myosin-X and fascin but independent of Cdc42 and VASP. Interestingly, in the presence of an actin polymerization inhibitor, namely, cytochalasin D, latrunculin A, or jasplakinolide, STS treatment induced excess tubulin polymerization, which resulted in the formation of many extra-long microtubule (MT)-based protrusions toward the outside of the cell. The unique MT-based protrusions were thick and linear compared with the STS-induced filaments or stationary filopodia. These protrusions, which were composed of microtubules, have been scarcely observed in cultured non-neuronal cells. Taken together, our findings revealed that STS-sensitive kinases are essential for the maintenance of normal cell morphology, and a common unidentified molecular mechanism is involved in the formation of the following two different types of protrusions: actin-based filaments and MT-based shafts.

DOI PubMed

Intercellular bridges for cell-cell communication

Takayuki Kohno

Tumor Research 45,67-71 2010 [Refereed]

Authorship： Lead author , Last author , Corresponding author

DOI

Attenuation of cell motility observed with high doses of sphingosine 1-phosphate or phosphorylated FTY720 involves RGS2 through its interactions with the receptor S1P

Takayuki Kohno, Yasuyuki Igarashi

GENES TO CELLS ( BLACKWELL PUBLISHING ) 13 ( 7 ) 747 - 757 2008.07 [Refereed]

Authorship： Lead author

Sphingosine 1-phosphate (S1P) stimulation enhances cell motility via the G-protein coupled S1P receptor S1P1. This ligand-induced, receptor-mediated cell motility follows a typical bell-shaped dose-response curve, that is, stimulation with low concentrations of S1P enhances cell motility, whereas excess ligand stimulation does not enhance it. So far, the attenuation of the response at higher ligand concentrations has not been explained. We report here that S1P1 interacts with the regulator of G protein signaling (RGS)-2 protein, which is a GTPase-activating protein (GAP) for heterotrimeric G proteins, in a concentration dependent manner. The RGS2-S1P1 complex dissociated at higher ligand concentrations, yet it was unaffected at low concentrations, suggesting that the dissociated RGS2 is involved in the concurrent decrease of cell motility. In RGS2 knockdown cells, the decrease of cell motility induced by high ligand concentrations was rescued. S1P1 internalization was not implicated in the attenuation of the response. Similar results were observed upon stimulation with the phosphorylated form of FTY720 (FTYP), which is an S1P1 agonist. In conclusion, the suppressed response in cell motility induced by excess S1P or FTYP via S1P1 is regulated by RGS2 functioning through a mechanism that is independent of S1P1 internalization.

DOI PubMed

Protein-tyrosine kinase CAK beta/PYK2 is activated by binding Ca2+/calmodulin to FERM F2 alpha 2 helix and thus forming its dimer

Takayuki Kohno, Eiko Matsuda, Hiroko Sasaki, Terukatsu Sasaki

BIOCHEMICAL JOURNAL ( PORTLAND PRESS LTD ) 410 513 - 523 2008.03 [Refereed]

Authorship： Lead author

CAK beta (cell adhesion kinase beta)/PYK2 (proline-rich tyrosine kinase 2) is the second protein-tyrosine kinase of the FAK (focal adhesion kinase) subfamily. It is different from FAK in that it is activated following an increase in cytoplasmic free Ca2+. In the present study we have investigated how Ca2+ activates CAK beta/PYK2. Calmodulin-agarose bound CAK beta/PYK2, but not FAK, in the presence of CaCl2. An alpha-helix (F2-alpha 2) present in the FERM (band four-point-one, ezrin, radixin, moesin homology) F2 subdomain of CAK beta/PYK2 was the binding site of Ca2+/calmodulin; a mutant of this region, L176A/Q177A (LQ/AA) CAK beta/PYK2, bound to Ca2+/calmodulin much less than the wild-type. CAK beta/PYK2 is known to be prominently tyrosine phosphorylated when overexpressed from cDNA. The enhanced tyrosine phosphorylation was inhibited by W7, an inhibitor of calmodulin, and by a cell-permeable Ca2+ chelator and was almost defective in the LQ/AA-mutant CAK beta/PYK2. CAK beta/PYK2 formed a homodimer on binding of Ca2+/calmodulin, which might then induce a conformational change of the kinase, resulting in transphosphorylation within the dimer. The dimer was formed at a free-Ca2+ concentration of 8-12 mu M and was stable at 500 nM Ca2+, but dissociated to a monomer in a Ca2+-free buffer. The dimer formation of CAK beta/PYK2 FERM domain was partially defective in the LQ/AA-mutant FERM domain and was blocked by W7 and by a synthetic peptide with amino acids 168-188 of CAK beta/PYK2, but not by a peptide with its LQ/AA-mutant sequence. It is known that the F2-alpha 2 helix is found immediately adjacent to a hydrophobic pocket in the FERM F2 lobe, which locks, in the autoinhibited FAK, the Globe of the kinase domain. Our results indicate that Ca2+/calmodulin binding to the FERM F2-alpha 2 helix of CAK beta/PYK2 releases its kinase domain from autoinhibition by forming a dimer.

DOI PubMed

Roles for N-glycosylation in the dynamics of Edg-1/S1P1 in sphingosine 1-phosphate-stimulated cells

T Kohno, Y Igarashi

GLYCOCONJUGATE JOURNAL ( SPRINGER ) 21 ( 8-9 ) 497 - 501 2004 [Refereed]

Authorship： Lead author

Sphingosine 1-phosphate (Sph-1-P) is a bioactive lipid mediator released from activated platelets. To date, 5 seven-transmembrane-spanning receptors, Edg-1/S1P1, Edg-3/S1P3, Edg-5/S1P2, Edg-6/S1P4 and Edg-8/S1P5, have been identified as specific Sph-1-P receptors. Our recent novel studies established that Edg-1/S1P1 is glycosylated in its N-terminal extracellular portion and further identified the specific glycosylation site as asparagine 30. We also demonstrated that the structure of the N-terminal ectodomain of Edg-1/S1P1 affects both its transport to the cell surface and the N-glycosylation process. These studies revealed a possible regulatory role for the N-glycan on Edg-1/S1P1 in the dynamics of the receptor, such as its lateral and internal movements within the membrane, in ligand-stimulated mammalian cells.

DOI PubMed

Truncation of the N-terminal ectodomain has implications in the N-glycosylation and transport to the cell surface of Edg-1/S1P1 receptor

T Kohno, Y Igarashi

JOURNAL OF BIOCHEMISTRY ( JAPANESE BIOCHEMICAL SOC ) 134 ( 5 ) 667 - 673 2003.11 [Refereed]

Authorship： Lead author

The endothelial cell-expressed sphingosine I-phosphate receptors Edg-1/S1P1 and Edg-3/S1P3 have been implicated in various physiological events such as the regulation of angiogenesis. Since there is an excess of a ligand constitutively in blood, these receptors may have some mechanism(s) avoiding overstimulation. In this study, we found that the N-terminal ectodomains of Edg-1/S1P1 and Edg-3/S1P3 were truncated in overexpressing cells. The truncated form of Edg-1/S1P1 expressed on the cell surface had undergone complex-type oligosaccharide modification at the Golgi. A deletion mutant lacking the N-terminal processing domain of Edg-1/S1P1 accumulated in the endoplasmic reticulum, and was not expressed on the cell surface. When a basic amino acid residue was introduced at the cleavage site of Edg-1/S1P1, the molecular weight of the glycosylated protein was greater in the mutant compared to the wild type, due to the bound oligosaccharide. These results demonstrated that the structure of the N-terminal ectodomain of Edg-1/S1P1 affects both its transport to the cell surface and the N-glycosylation process. Ectodomain shedding of many membrane proteins has been implicated in various diseases. Therefore, N-terminal processing of Edg-1/S1P1 and Edg-3/S1P3 might play roles in endothelial cell functions.

DOI PubMed

Sphingosine 1-phosphate promotes cell migration through the activation of Cdc42 in Edg-6/S1P4-expressing cells

T Kohno, H Matsuyuki, Y Inagaki, Y Igarashi

GENES TO CELLS ( BLACKWELL PUBLISHING LTD ) 8 ( 8 ) 685 - 697 2003.08 [Refereed]

Authorship： Lead author

Background: Sphingosine 1-phosphate (Sph-1-P) is a bioactive lipid mediator released from activated platelets, which regulates diverse signal transduction pathways via cell surface receptors. Recent studies have revealed that the seven-transmembrane-spanning receptors, Edg-1, Edg-3, Edg-5, Edg-6 and Edg-8 are specific Sph-1-P receptors. Northern blot analysis has demonstrated that Edg-6 is expressed in lymphocyte-containing tissues such as spleen and lung. Little is known about the molecular mechanisms of Edg-6 functions, probably because of the difficulties in expressing Edg-6 on the cell surface. Results: Here, our studies revealed that N-terminal FLAG-tagged Edg-6 or Edg-6-GFP fusion protein was expressed in the endoplasmic reticulum, but was not expressed on the cell surface. On the other hand, C-terminally tagged Edg-6 or both N-terminally and C-terminally tagged Edg-6 was able to localize to the cell surface. Using these cells, we found that Sph-1-P induced cell migration through cell surface-expressed Edg-6 in a pertussis toxin-sensitive manner. This motility was mediated through the activation of a member of the Rho family of small GTPases, Cdc42. Conclusion: These results support a role for Sph-1-P signalling via Edg-6 in the pathways involved in cell motility.

DOI PubMed

N-glycans of sphingosine 1-phosphate receptor Edg-1 regulate ligand-induced receptor internalization

T Kohno, A Wada, Y Igarashi

FASEB JOURNAL ( FEDERATION AMER SOC EXP BIOL ) 16 ( 9 ) 983 - 992 2002.07 [Refereed]

Authorship： Lead author

Endothelial differentiation gene-1 product (Edg-1) is a G-protein-coupled receptor (GPCR) for the platelet derived bioactive lipid mediator sphingosine 1-phosphate (Sph-1-P). Recent studies have shown that in response to Sph-1-P, Edg-1 mediates various signaling pathways through downstream signaling molecules, such as MAP kinase and calcium, via heterotrimeric G-proteins. We found for the first time that Edg-1 is glycosylated in its amino-terminal extracellular portion, and further identified the specific glycosylation site as asparagine 30 by creating a nonglycosylated mutant of Edg-1 (N30D-Edg-1) and transfecting it into cell lines. The nonglycosylated mutant receptors, resembling their wild-type controls, were predominantly expressed in the plasma membrane. Although there was no difference in ligand binding ability and ligand-induced MAP kinase activation in the wild-type and mutant receptors, nonglycosylated Edg-1 was much less responsive for ligand-induced internalization. Unlike the wild-type receptor, which was associated with the caveolae, nonglycosylated N30D-Edg-1 was dispersed broadly in the membrane fractions separated by sucrose density gradient centrifugation, suggesting that internalization and microdomain localization of N-glycosylated Edg-1 might be related. Although the precise molecular mechanism of the internalization of the N-glycosylated Edg-1 localized in the microdomain remains to be examined, the present study suggested that the presence of N-linked glycan in the receptor may play a regulatory role in the receptor dynamics in ligand-stimulated mammalian cells.

DOI PubMed

[N-glycosylation of sphingosine 1-phosphate receptor, Edg-1, and its role on receptor internalization through membrane microdomain].

Takayuki Kohno, Yasuyuki Igarashi

Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme 47(4)503-8 2002.03 [Refereed]

Authorship： Lead author

PubMed

The roles of tight junction protein cingulin in human endometrioid endometrial cancer.

Arisa Kura, Kimihito Saito, Takumi Konno, Takayuki Kohno, Hiroshi Shimada, Tadahi Okada, Soshi Nishida, Daichi Ishii, Motoki Matsuura, Tsuyoshi Saito, Takashi Kojima

Tissue barriers 2361976 - 2361976 2024.06 [Refereed] [International journal]

The bicellular tight junction molecule cingulin (CGN) binds to microtubules in centrosomes. Furthermore, CGN contributes to the tricellular tight junction (tTJ) proteins lipolysis-stimulated lipoprotein receptor (LSR) and tricellulin (TRIC). CGN as well as LSR decreased during the malignancy of endometrioid endometrial cancer (EEC). Although tTJ protein LSR is involved in the malignancy of some cancers, including EEC, the role of CGN is unknown. In this study, we investigated the roles of CGN with tTJ proteins in human EEC cells by using the CGN-overexpressing EEC cell line Sawano. In 2D cultures, CGN was colocalized with LSR and TRIC at tTJ or at γ-tubulin-positive centrosomes. In immunoprecipitation with CGN antibodies, CGN directly bound to LSR, TRIC, and β-tubulin. Knockdown of CGN by the siRNA decreased the epithelial barrier and enhanced cell proliferation, migration and invasion, as well as knockdown of LSR. In the Sawano cells cocultured with normal human endometrial stromal cells, knockdown of CGN decreased expression of LSR and TRIC via MAPK and AMPK pathways. In 2.5D cultures, knockdown of CGN induced the formation of abnormal cysts and increased the permeability of FD-4 to the lumen. In 2D and 2.5D cultures, treatment with β-estradiol with or without EGF or TGF-β decreased CGN expression and the epithelial permeability barrier and enhanced cell migration, and pretreatment with EW7197+AG1478, U0126 or an anti-IL-6 antibody prevented this. In conclusion, CGN, with tTJ proteins might suppress the malignancy of human EEC and its complex proteins are sensitive to estrogen and growth factors derived from stromal cells.

DOI PubMed

The Roles and Regulatory Mechanisms of Tight Junction Protein Cingulin and Transcription Factor Forkhead Box Protein O1 in Human Lung Adenocarcinoma A549 Cells and Normal Lung Epithelial Cells

Daichi Ishii, Yuma Shindo, Wataru Arai, Takumi Konno, Takayuki Kohno, Kazuya Honda, Masahiro Miyajima, Atsushi Watanabe, Takashi Kojima

International Journal of Molecular Sciences 2024.01 [Refereed]

DOI

The interplay between the epithelial permeability barrier, cell migration and mitochondrial metabolism of growth factors and their inhibitors in a human endometrial carcinoma cell line.

Takumi Konno, Takayuki Kohno, Kikuchi S, Kura A, Saito K, Okada T, Shimada H, Yamazaki Y, Sugiyama T, Matsuura M, Ohsaki Y, Saito T, Kojima T

Tissue barriers 2024.01 [Refereed]

It is known that there are abnormalities of tight junction functions, cell migration and mitochondrial metabolism in human endometriosis and endometrial carcinoma. In this study, we investigated the effects of growth factors and their inhibitors on the epithelial permeability barrier, cell migration and mitochondrial metabolism in 2D and 2.5D cultures of human endometrioid endometrial carcinoma Sawano cells. We also investigated the changes of bicellular and tricellular tight junction molecules and ciliogenesis induced by these inhibitors. The growth factors TGF-β and EGF affected the epithelial permeability barrier, cell migration and expression of bicellular and tricellular tight junction molecules in 2D and 2.5D cultures of Sawano cells. EW-7197 (a TGF-β receptor inhibitor), AG1478 (an EGFR inhibitor) and SP600125 (a JNK inhibitor) affected the epithelial permeability barrier, cell migration and mitochondrial metabolism and prevented the changes induced by TGF-β and EGF in 2D and 2.5D cultures. EW-7197 and AG1478 induced ciliogenesis in 2.5D cultures. In conclusion, TGF-β and EGF promoted the malignancy of endometrial cancer via interplay among the epithelial permeability barrier, cell migration and mitochondrial metabolism. EW-7197 and AG1478 may be useful as novel therapeutic treatments options for endometrial cancer.

DOI PubMed

Oxygen–Glucose Deprivation Decreases the Motility and Length of Axonal Mitochondria in Cultured Dorsal Root Ganglion Cells of Rats

Shin Kikuchi, Takayuki Kohno, Takashi Kojima, Haruyuki Tatsumi, Yuki Ohsaki, Takafumi Ninomiya

Cellular and Molecular Neurobiology 2023.04 [Refereed]

DOI

Downregulation of angulin-1/LSR induces malignancy via upregulation of EGF-dependent claudin-2 and TGF-β-dependent cell metabolism in human lung adenocarcinoma A549 cells.

Wataru Arai, Takumi Konno, Takayuki Kohno, Yuki Kodera, Mitsuhiro Tsujiwaki, Yuma Shindo, Hirofumi Chiba, Masahiro Miyajima, Yuji Sakuma, Atsushi Watanabe, Takashi Kojima

Oncotarget 14 261 - 275 2023.03 [Refereed] [International journal]

Abnormal expression of bicellular tight junction claudins, including claudin-2 are observed during carcinogenesis in human lung adenocarcinoma. However, little is known about the role of tricellular tight junction molecule angulin-1/lipolysis-stimulated lipoprotein receptor (LSR). In the lung adenocarcinoma tissues examined in the present study, expression of claudin-2 was higher than in normal lung tissues, while angulin-1/LSR was poorly or faintly expressed. We investigated how loss of angulin-1/LSR affects the malignancy of lung adenocarcinoma cell line A549 and normal human lung epithelial (HLE) cells. The EGF receptor tyrosine kinase inhibitor AG1478 prevented the increase of claudin-2 expression induced by EGF in A549 cells. Knockdown of LSR induced expression of claudin-2 at the protein and mRNA levels and AG1478 prevented the upregulation of claudin-2 in A549 cells. Knockdown of LSR induced cell proliferation, cell migration and cell metabolism in A549 cells. Knockdown of claudin-2 inhibited the cell proliferation but did not affect the cell migration or cell metabolism of A549 cells. The TGF-β type I receptor inhibitor EW-7197 prevented the decrease of LSR and claudin-2 induced by TGF-β1 in A549 cells and 2D culture of normal HLE cells. EW-7197 prevented the increase of cell migration and cell metabolism induced by TGF-β1 in A549 cells. EW-7197 prevented the increase of epithelial permeability of FITC-4kD dextran induced by TGF-β1 in 2.5D culture of normal HLE cells. In conclusion, downregulation of angulin-1/LSR induces malignancy via EGF-dependent claudin-2 and TGF-β-dependent cell metabolism in human lung adenocarcinoma.

DOI PubMed

LSR antibody promotes apoptosis and disrupts epithelial barriers via signal pathways in endometrial cancer.

Kimihito Saito, Takumi Konno, Takayuki Kohno, Hiroshi Shimada, Motoki Matsuura, Tadahi Okada, Arisa Kura, Daichi Ishii, Masuo Kondoh, Tsuyoshi Saito, Takashi Kojima

Tissue barriers 2106113 - 2106113 2022.07 [Refereed] [International journal]

Lipolysis-stimulated lipoprotein receptor (LSR), a lipid metabolism-related factor localized in tricellular tight junctions (tTJs), plays an important role in maintaining the epithelial barrier. LSR is highly expressed in well-differentiated endometrial endometrioid carcinoma (EEC), and its expression decreases during malignancy. Angubindin-1, a novel LSR ligand peptide, regulates tTJs without cytotoxicity, enhances paracellular permeability, and regulates epithelial barrier via c-Jun N-terminal kinase (JNK)/cofilin. In this study, we investigated the immune-modulatory roles of an anti-LSR antibody in the treatment of EEC in vitro compared to those of angubindin-1. We prepared an antibody against the extracellular N-terminal domain of human LSR (LSR-N-ab) and angubindin-1. EEC cell-line Sawano cells in 2D and 2.5D cultures were treated with 100 μg/ml LSR-N-ab or 2.5 μg/ml angubindin-1 with or without protein tyrosine kinase 2β inhibitor PF431396 (PF43) and JNK inhibitor SP600125 (SP60) at 10 μM. Treatment with LSR-N-ab and angubindin-1 decreased LSR at the membranes of tTJs and the activity of phosphorylated LSR and phosphorylated cofilin in 2D culture. Treatment with LSR-N-ab and angubindin-1 decreased the epithelial barrier measured as TEER values in 2D culture and enhanced the epithelial permeability of FD-4 in 2.5D culture. Treatment with LSR-N-ab, but not angubindin-1, induced apoptosis in 2D culture. Pretreatment with PF43 and SP60 prevented all the changes induced by treatment with LSR-N-ab and angubindin-1. Treatment with LSR-N-ab and angubindin-1 enhanced the cell metabolism measured as the mitochondrial respiration levels in 2D culture. LSR-N-ab and angubindin-1 may be useful for therapy of human EEC via enhanced apoptosis or drug absorption.

DOI PubMed

Inhibition of HDAC and Signal Transduction Pathways Induces Tight Junctions and Promotes Differentiation in p63-Positive Salivary Duct Adenocarcinoma

Masaya Nakano, Kizuku Ohwada, Yuma Shindo, Takumi Konno, Takayuki Kohno, Shin Kikuchi, Mitsuhiro Tsujiwaki, Daichi Ishii, Soshi Nishida, Takuya Kakuki, Kazufumi Obata, Ryo Miyata, Makoto Kurose, Atsushi Kondoh, Kenichi Takano, Takashi Kojima

Cancers 14 ( 11 ) 2022.05 [Refereed] [International journal]

BACKGROUND: The p53 family p63 is essential for the proliferation and differentiation of various epithelial basal cells. It is overexpressed in several cancers, including salivary gland neoplasia. Histone deacetylases (HDACs) are thought to play a crucial role in carcinogenesis, and HDAC inhibitors downregulate p63 expression in cancers. METHODS: In the present study, to investigate the roles and regulation of p63 in salivary duct adenocarcinoma (SDC), human SDC cell line A253 was transfected with siRNA-p63 or treated with the HDAC inhibitors trichostatin A (TSA) and quisinostat (JNJ-26481585). RESULTS: In a DNA array, the knockdown of p63 markedly induced mRNAs of the tight junction (TJ) proteins cingulin (CGN) and zonula occuludin-3 (ZO-3). The knockdown of p63 resulted in the recruitment of the TJ proteins, the angulin-1/lipolysis-stimulated lipoprotein receptor (LSR), occludin (OCLN), CGN, and ZO-3 at the membranes, preventing cell proliferation, and leading to increased cell metabolism. Treatment with HDAC inhibitors downregulated the expression of p63, induced TJ structures, recruited the TJ proteins, increased the epithelial barrier function, and prevented cell proliferation and migration. CONCLUSIONS: p63 is not only a diagnostic marker of salivary gland neoplasia, but it also promotes the malignancy. Inhibition of HDAC and signal transduction pathways is, therefore, useful in therapy for p63-positive SDC cells.

DOI PubMed

FOXO3/TGF-β signal-dependent ciliogenesis and cell functions during differentiation of temperature-sensitive mouse cochlear precursor hair cells.

Takuya Kakuki, Takayuki Kohno, Soshi Nishida, Takumi Konno, Shin Kikuchi, Kizuku Ohwada, Masaya Nakano, Mitsuki Tezuka, Kenichi Takano, Takashi Kojima

Histochemistry and cell biology 157 ( 4 ) 415 - 426 2022.04 [Refereed] [International journal]

The transcription factor FOXO3 is necessary to preserve cochlear hair cells. Growth factors, including TGF-β, closely contribute to cochlear hair cell regeneration. In the present study, to investigate the roles of FOXO3 in the ciliogenesis and cell functions of cochlear hair cells, UB/OC-2 temperature-sensitive mouse cochlear precursor hair cells were treated with TGF-β receptor type 1 inhibitor EW-7197 or EGF receptor inhibitor AG-1478 after transfection with or without siRNA-FOXO3a. GeneChip analysis revealed that treatment with EW-7197 increased Foxo3 genes and decreased genes of Smads. During cell differentiation, treatment with EW-7197 or AG-1478 induced an increase in length of cilia-like structures that were positive for acetylated tubulin and inhibited cell migration. Treatment with EW-7197 also increased cell metabolism measured as mitochondrial basal respiration (oxygen consumption rate). The effects of EW-7197 were stronger than those of AG-1478. Knockdown of FOXO3 prevented the growth of cilia-like structures induced by EW-7197 or AG-1478 and induced cell migration under treatment with EW-7197. No change of the epithelial cell polarity molecule PAR3 was observed with any treatment. Treatment with the antimicrobial agent amikacin prevented the growth of cilia-like structures induced by EW-7197 and induced apoptosis. Pretreatment with the glucocorticoid dexamethasone inhibited the apoptosis induced by amikacin. This in vitro model of mouse cochlear hair cells suggests that FOXO3/TGF-β signaling plays a crucial role in ciliogenesis and cell functions during differentiation of cochlear hair cells. This model is useful for analysis of the mechanisms of hearing loss and to find therapeutic agents to prevent it.

DOI PubMed

The Roles of Tricellular Tight Junction Protein Angulin-1/Lipolysis-Stimulated Lipoprotein Receptor (LSR) in Endometriosis and Endometrioid-Endometrial Carcinoma.

Hiroshi Shimada, Takayuki Kohno, Takumi Konno, Tadahi Okada, Kimihito Saito, Yuma Shindo, Shin Kikuchi, Mitsuhiro Tsujiwaki, Marie Ogawa, Motoki Matsuura, Tsuyoshi Saito, Takashi Kojima

Cancers 13 ( 24 ) 2021.12 [Refereed] [International journal]

Tight junction proteins play roles beyond permeability barriers functions and control cell proliferation and differentiation. The relation between tight junctions and the signal transduction pathways affects cell growth, invasion and migration. Abnormality of tight junction proteins closely contributes to epithelial mesenchymal transition (EMT) and malignancy of various cancers. Angulin-1/lipolysis-stimulated lipoprotein receptor (LSR) forms tricellular contacts that has a barrier function. Downregulation of angulin-1/LSR correlates with the malignancy in various cancers, including endometrioid-endometrial carcinoma (EEC). These alterations have been shown to link to not only multiple signaling pathways such as Hippo/YAP, HDAC, AMPK, but also cell metabolism in ECC cell line Sawano. Moreover, loss of angulin-1/LSR upregulates claudin-1, and loss of apoptosis stimulating p53 protein 2 (ASPP2) downregulates angulin-1/LSR. Angulin-1/LSR and ASPP2 concentrate at both midbody and centrosome in cytokinesis. In EEC tissues, angulin-1/LSR and ASPP2 are reduced and claudin-2 is overexpressed during malignancy, while in the tissues of endometriosis changes in localization of angulin-1/LSR and claudin-2 are seen. This review highlights how downregulation of angulin-1/LSR promotes development of endometriosis and EEC and discusses about the roles of angulin-1/LSR and its related proteins, including claudins and ASPP2.

DOI PubMed

Dysfunction of epithelial permeability barrier induced by HMGB1 in 2.5D cultures of human epithelial cells

Takashi Kojima, Yuma Shindo, Takumi Konno, Yuki Kodera, Wataru Arai, Maki Miyakawa, Kizuku Ohwada, Hiroki Tanaka, Mitsuhiro Tsujiwaki, Yuji Sakuma, Shin Kikuchi, Tsuyoshi Ohkuni, Kenichi Takano, Atsushi Watanabe, Takayuki Kohno

Tissue Barriers ( Informa {UK} Limited ) 10 ( 2 ) 1972760 - 1972760 2021.09 [Refereed] [International journal]

Authorship： Last author

Airway and intestinal epithelial permeability barriers are crucial in epithelial homeostasis. High mobility group box 1 (HMGB1), increased by various stimuli, is involved in the induction of airway inflammation, as well as the pathogenesis of inflammatory bowel disease. HMGB1 enhances epithelial hyperpermeability. Two-and-a-half dimensional (2.5D) culture assays are experimentally convenient and induce cells to form a more physiological tissue architecture than 2D culture assays for molecular transfer mechanism analysis. In 2.5D culture, treatment with HMGB1 induced permeability of FITC-dextran into the lumen formed by human lung, nasal and intestinal epithelial cells. The tricellular tight junction molecule angulin-1/LSR is responsible for the epithelial permeability barrier at tricellular contacts and contributes to various human airway and intestinal inflammatory diseases. In this review, we indicate the mechanisms including angulin-1/LSR and multiple signaling in dysfunction of the epithelial permeability barrier induced by HMGB1 in 2.5D culture of human airway and intestinal epithelial cells.

DOI PubMed

Effects of HMGB1 on Tricellular Tight Junctions via TGF-β Signaling in Human Nasal Epithelial Cells

Kizuku Ohwada, Takumi Konno, Takayuki Kohno, Masaya Nakano, Tsuyoshi Ohkuni, Ryo Miyata, Takuya Kakuki, Masuo Kondoh, Kenichi Takano, Takashi Kojima

International Journal of Molecular Sciences ( {MDPI} {AG} ) 22 ( 16 ) 8390 - 8390 2021.08 [Refereed] [International journal]

The airway epithelium of the human nasal mucosa acts as a physical barrier that protects against inhaled substances and pathogens via bicellular and tricellular tight junctions (bTJs and tTJs) including claudins, angulin-1/LSR and tricellulin. High mobility group box-1 (HMGB1) increased by TGF-β1 is involved in the induction of nasal inflammation and injury in patients with allergic rhinitis, chronic rhinosinusitis, and eosinophilic chronic rhinosinusitis. However, the detailed mechanisms by which this occurs remain unknown. In the present study, to investigate how HMGB1 affects the barrier of normal human nasal epithelial cells, 2D and 2.5D Matrigel culture of primary cultured human nasal epithelial cells were pretreated with TGF-β type I receptor kinase inhibitor EW-7197 before treatment with HMGB1. Knockdown of angulin-1/LSR downregulated the epithelial barrier. Treatment with EW-7197 decreased angulin-1/LSR and concentrated the expression at tTJs from bTJs and increased the epithelial barrier. Treatment with a binder to angulin-1/LSR angubindin-1 decreased angulin-1/LSR and the epithelial barrier. Treatment with HMGB1 decreased angulin-1/LSR and the epithelial barrier. In 2.5D Matrigel culture, treatment with HMGB1 induced permeability of FITC-dextran (FD-4) into the lumen. Pretreatment with EW-7197 prevented the effects of HMGB1. HMGB1 disrupted the angulin-1/LSR-dependent epithelial permeability barriers of HNECs via TGF-β signaling in HNECs.

DOI PubMed

Effects of histone deacetylase inhibitors Tricostatin A and Quisinostat on tight junction proteins of human lung adenocarcinoma A549 cells and normal lung epithelial cells

Yuma Shindo, Wataru Arai, Takumi Konno, Takayuki Kohno, Yuki Kodera, Hirofumi Chiba, Masahiro Miyajima, Yuji Sakuma, Atsushi Watanabe, Takashi Kojima

Histochemistry and Cell Biology ( Springer Science and Business Media LLC ) 155 ( 6 ) 637 - 653 2021.06 [Refereed] [International journal]

Histone deacetylase (HDAC) inhibitors have a potential therapeutic role for non-small cell lung cancer (NSCLC). However, more preclinical studies of HDAC inhibitors in NSCLC and normal lung epithelial cells are required to evaluate their antitumor activities and mechanisms. The bicellular tight junction molecule claudin-2 (CLDN-2) is highly expressed in lung adenocarcinoma tissues and increase the proliferation of adenocarcinoma cells. Downregulation of the tricellular tight junction molecule angulin-1/LSR induces malignancy via EGF-dependent CLDN-2 and TGF-β-dependent cellular metabolism in human lung adenocarcinoma cells. In the present study, to investigate the detailed mechanisms of the antitumor activities of HDAC inhibitors in lung adenocarcinoma, human lung adenocarcinoma A549 cells and normal lung epithelial cells were treated with the HDAC inibitors Trichostatin A (TSA) and Quisinostat (JNJ-2648158) with or without TGF-β. Both HDAC inhibitors increased anguin-1/LSR, decrease CLDN-2, promoted G1 arrest and prevented the migration of A549 cells. Furthermore, TSA but not Quisinostat with or without TGF-β induced cellular metabolism indicated as the mitochondrial respiration measured using the oxygen consumption rate. In normal human lung epithelial cells, treatment with TSA and Quisinostat increased expression of LSR and CLDN-2 and decreased that of CLDN-1 with or without TGF-β in 2D culture. Quisinostat but not TSA with TGF-β increased CLDN-7 expression in 2D culture. Both HDAC inhibitors prevented disruption of the epithelial barrier measured as the permeability of FD-4 induced by TGF-β in 2.5D culture. TSA and Quisinostat have potential for use in therapy for lung adenocarcinoma via changes in the expression of angulin-1/LSR and CLDN-2.

DOI PubMed

Pyk2 inhibitor prevents epithelial hyperpermeability induced by HMGB1 and inflammatory cytokines in Caco-2 cells

Takumi Konno, Takayuki Kohno, Maki Miyakawa, Hiroki Tanaka, Takashi Kojima

Tissue Barriers ( Informa UK Limited ) 9 ( 2 ) 1890526 - 1890526 2021.04 [Refereed] [International journal]

The non-receptor protein tyrosine kinase 2β (Pyk2) phosphorylated tricellular tight junction (tTJ) molecules angulin-1/LSR and tricellulin (TRIC) and the inhibitor PF-431396 (PF43) suppress angulin-1/LSR and TRIC recruitment to tTJs. The disruption of the intestinal epithelial barrier by high mobility group box 1 (HMGB1) and the inflammatory cytokines TNFα and IFNγ contributes to downregulation of angulin-1/LSR and TRIC in 2.5D culture of Caco-2 cells as a novel model of inflammatory bowel disease (IBD). In the present study, to investigate the roles of Pyk2 phosphorylated angulin-1/LSR and TRIC in the intestinal epithelial barrier, 2D and 2.5D cultures of Caco-2 cells were treated with the Pyk2 inhibitor PF-43 with or without HMGB1, inflammatory cytokines TNFα and IFNγ. Treatment with PF-43 increased expression of angulin-1/LSR, phosphorylated AMPK and phosphorylated MAPK and decreased that of phosphorylated JNK, with upregulation of the epithelial barrier and cellular metabolism measured as basal oxygen consumption rate (OCR) and ATP production in 2D culture. Treatment with PF-43 prevented the downregulation of the epithelial barrier by HMGB1 and inflammatory cytokines in 2D culture. Treatment with PF-43 prevented the epithelial hyperpermeability induced by HMGB1 and inflammatory cytokines in 2.5D culture. In 2.5D culture, treatment with PF-43 inhibited the decreases of angulin-1/LSR, TRIC, pJNK, pAMPK and pMAPK induced by HMGB1 and the inflammatory cytokines. Treatment with PF-43 inhibited in part the induced phosphorylation of the serine of angulin-1/LSR and TRIC. Pyk2 inhibitor PF-43 may have potential for use in therapy for IBD via its actions with regard to phosphorylated tTJs and cellular metabolism.

DOI PubMed

HDAC inhibitors suppress the proliferation, migration and invasiveness of human head and neck squamous cell carcinoma cells via p63‑mediated tight junction molecules and p21‑mediated growth arrest

Akito Kakiuchi, Takuya Kakuki, Kizuku Ohwada, Makoto Kurose, Atsushi Kondoh, Kazufumi Obata, Kazuaki Nomura, Ryo Miyata, Yakuto Kaneko, Takumi Konno, Takayuki Kohno, Tetsuo Himi, Ken-Ichi Takano, Takashi Kojima

Oncology Reports ( Spandidos Publications ) 45 ( 4 ) 2021.03 [Refereed] [International journal]

In human head and neck squamous cell carcinoma (HNSCC), the invasion and metastatic properties of cancer cells are promoted by junctional adhesion molecule‑A (JAM‑A) and claudin‑1; these are epithelial tight junction molecules regulated by histone deacetylases (HDACs) and transcription factor p63. HDAC expression is reportedly upregulated in HNSCC, and HDAC inhibitors suppress cancer cell proliferation by initiating proliferative arrest or apoptosis. However, little is known of the anti‑cancer mechanisms of HDAC inhibitors in HNSCC. Thus, in the present study, the HNSCC Detroit 562 cell line and primary cultured HNSCC cells were treated with HDAC inhibitors to investigate their effects in HNSCC. Higher expression of p63, HDAC1, JAM‑A and claudin‑1 was observed in HNSCC tissues compared with the adjacent dysplastic regions. In Detroit 562 cells, treatment with trichostatin A (TSA), an inhibitor of HDAC1 and 6, downregulated the expression of p63, JAM‑A and claudin‑1, and upregulated that of acetylated tubulin; conversely, p63 knockdown resulted in the downregulation of JAM‑A and claudin‑1. Collectively, inhibiting HDAC suppressed the migration and invasiveness of cancer cells. In addition, treatment with TSA suppressed cancer cell proliferation via G2/M arrest, as well as upregulating p21 and downregulating cyclin D1 expression. TSA also downregulated the expression of epidermal growth factor receptor (EGFR) and phospho‑ERK1/2. p63 knockdown and treatment with an EGFR inhibitor induced G1 arrest and downregulated EGFR and phospho‑ERK1/2 levels, respectively. HDAC inhibition also suppressed the migration and invasiveness of primary cultured HNSCC cells. Collectively, the results of the present study indicate that HDAC inhibitors suppress the proliferation, migration and invasiveness of HNSCC by downregulating the p63‑mediated tight junction molecules JAM‑A and claudin‑1, and inducing p63 or p21‑mediated growth arrest.

DOI PubMed

Increase in Epithelial Permeability and Cell Metabolism by High Mobility Group Box 1, Inflammatory Cytokines and TPEN in Caco-2 Cells as a Novel Model of Inflammatory Bowel Disease.

Maki Miyakawa, Takumi Konno, Takayuki Kohno, Shin Kikuchi, Hiroki Tanaka, Takashi Kojima

International journal of molecular sciences 21 ( 22 ) 2020.11 [Refereed] [International journal]

High mobility group box 1 protein (HMGB1) is involved in the pathogenesis of inflammatory bowel disease (IBD). Patients with IBD develop zinc deficiency. However, the detailed roles of HMGB1 and zinc deficiency in the intestinal epithelial barrier and cellular metabolism of IBD remain unknown. In the present study, Caco-2 cells in 2D culture and 2.5D Matrigel culture were pretreated with transforming growth factor-β (TGF-β) type 1 receptor kinase inhibitor EW-7197, epidermal growth factor receptor (EGFR) kinase inhibitor AG-1478 and a TNFα antibody before treatment with HMGB1 and inflammatory cytokines (TNFα and IFNγ). EW-7197, AG-1478 and the TNFα antibody prevented hyperpermeability induced by HMGB1 and inflammatory cytokines in 2.5D culture. HMGB1 affected cilia formation in 2.5D culture. EW-7197, AG-1478 and the TNFα antibody prevented the increase in cell metabolism induced by HMGB1 and inflammatory cytokines in 2D culture. Furthermore, ZnSO4 prevented the hyperpermeability induced by zinc chelator TPEN in 2.5D culture. ZnSO4 and TPEN induced cellular metabolism in 2D culture. The disruption of the epithelial barrier induced by HMGB1 and inflammatory cytokines contributed to TGF-β/EGF signaling in Caco-2 cells. The TNFα antibody and ZnSO4 as well as EW-7197 and AG-1478 may have potential for use in therapy for IBD.

DOI PubMed

Possibility of Targeting Claudin-2 in Therapy for Human Endometrioid Endometrial Carcinoma.

Tadahi Okada, Takumi Konno, Takayuki Kohno, Hiroshi Shimada, Kimihito Saito, Seiro Satohisa, Tsuyoshi Saito, Takashi Kojima

Reproductive sciences (Thousand Oaks, Calif.) 27 ( 11 ) 2092 - 2103 2020.11 [Refereed] [International journal]

Claudin-2 (CLDN-2) is a leaky-type tight junction protein, and its overexpression increases tumorigenesis of some types of cancer cells. In the present study, to examine the possibility of targeting CLDN-2 in the therapy for endometrioid endometrial adenocarcinoma, we investigated the regulation and role of CLDN-2 in endometriosis and endometrioid endometrial adenocarcinoma. In endometrioid endometrial adenocarcinoma tissues, marked upregulation of CLDN-2 was observed together with malignancy, while in endometriosis tissues, a change in the localization of CLDN-2 was observed. In cells of the endometrial adenocarcinoma cell line Sawano, which highly express CLDN-2, downregulation of CLDN-2 induced by the siRNA upregulated the epithelial barrier and inhibited cell migration. Furthermore, the downregulation of CLDN-2 affected the cell cycle and inhibited cell proliferation. In Sawano cells cultured with high-glucose medium, CLDN-2 expression was downregulated at the mRNA and protein levels. The high-glucose medium upregulated the epithelial barrier, cell proliferation, and migration, and inhibited cell invasion. The histone deacetylase (HDAC) inhibitor tricostatin A (TSA), which has antitumor effects, downregulated CLDN-2 expression, cell proliferation, invasion, and migration, and upregulated the epithelial barrier. The mitochondrial respiration level, an indicator of cancer metabolism, was downregulated by CLDN-2 knockdown and upregulated by the high-glucose condition. Taken together, these results indicated that overexpression of CLDN-2 closely contributed to the malignancy of endometrioid endometrial adenocarcinoma. Downregulation of CLDN-2 via the changes of the glucose concentration and treatment with HDAC inhibitors may be important in the therapy for endometrial cancer.

DOI PubMed

HMGB1 enhances epithelial permeability via p63/TGF-β signaling in lung and terminal bronchial epithelial cells.

Yuki Kodera, Takayuki Kohno, Takumi Konno, Wataru Arai, Mitsuhiro Tsujiwaki, Yuma Shindo, Hirofumi Chiba, Maki Miyakawa, Hiroki Tanaka, Yuji Sakuma, Atsushi Watanabe, Hiroki Takahashi, Takashi Kojima

Tissue barriers 8 ( 4 ) 1805997 - 1805997 2020.10 [Refereed] [International journal]

High mobility group box 1 (HMGB1) is involved in the induction of airway inflammation and injury in patients with chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis (IPF). HMGB1 increased by transforming growth factor-β1 (TGF-β1), impairs airway epithelial barrier function in the lung. In the present study, to investigate how HMGB1 affects the barrier of normal human lung epithelial (HLE) cells, monolayer cells (2D culture) and bronchial-like spheroid cells (2.5 D Matrigel culture), which have lumen formation, were pretreated with TGF-β type I receptor kinase inhibitor EW-7197 before treatment with HMGB1. In 2D culture, treatment with HMGB1 decreased expression of angulin-1/LSR, TRIC and CLDN-1, -4, -7 and increased that of CLDN-2. Pretreatment with EW-7197 prevented the changes of all tight junction molecules induced by HMGB1. In 2.5D Matrigel culture, treatment with HMGB1 induced permeability of FITC-dextran (FD-4) into the lumen, whereas pretreatment with EW-7197 prevented the hyperpermeability of FD-4 into the lumen caused by HMGB1. In 2.5D Matrigel culture, knockdown of transcription factor p63 prevented the hyperpermeability induced by HMGB1 as well as pretreatment with EW-7197. In the 2D culture of HLE cells with HMGB1, knockdown of p63 increased the level of angulin-1/LSR and CLDN-4, while pretreatment with EW-7197 enhanced the increase of CLDN-4 induced by knockdown of p63. Immunohistochemical analysis of IPF, CLDN-2, HMGB1 and p63 revealed that their levels were higher in the regenerative epithelium of the terminal bronchial region than in normal epithelium. HMGB1 induces epithelial permeability of HLE cells via p63/TGF-β signaling in normal lung and IPF.

DOI PubMed

ASPP2 suppression promotes malignancy via LSR and YAP in human endometrial cancer.

Takumi Konno, Takayuki Kohno, Tadahi Okada, Hiroshi Shimada, Seiro Satohisa, Shin Kikuchi, Tsuyoshi Saito, Takashi Kojima

Histochemistry and cell biology 154 ( 2 ) 197 - 213 2020.08 [Refereed] [International journal]

Apoptosis-stimulating p53 protein 2 (ASPP2) is an apoptosis inducer that acts via binding with p53 and epithelial polarity molecule PAR3. Lipolysis-stimulated lipoprotein receptor (LSR) is an important molecule at tricellular contacts, and loss of LSR promotes cell migration and invasion via Yes-associated protein (YAP) in human endometrial cancer cells. In the present study, to find how ASPP2 suppression promotes malignancy in human endometrial cancer, we investigated its mechanisms including the relationship with LSR. In endometriosis and endometrial cancers (G1 and G2), ASPP2 was observed as well as PAR3 and LSR in the subapical region. ASPP2 decreased in G3 endometrial cancer compared to G1. In human endometrial cancer cell line Sawano, ASPP2 was colocalized with LSR and tricellulin at tricellular contacts and binding to PAR3, LSR, and tricellulin in the confluent state. ASPP2 suppression promoted cell migration and invasion, decreased LSR expression, and induced expression of phosphorylated YAP, claudin-1, -4, and -7 as effectively as the loss of LSR. Knockdown of YAP prevented the upregulation of pYAP, cell migration and invasion induced by the ASPP2 suppression. Treatment with a specific antibody against ASPP2 downregulated ASPP2 and LSR, affected F-actin at tricellular contacts, upregulated expression of pYAP and claudin-1, and induced cell migration and invasion via YAP. In normal human endometrial epithelial cells, ASPP2 was in part colocalized with LSR at tricellular contacts and knockdown of ASPP2 or LSR induced expression of claudin-1 and claudin-4. ASPP2 suppression promoted cell invasion and migration via LSR and YAP in human endometrial cancer cells.

DOI PubMed

HMGB1-downregulated angulin-1/LSR induces epithelial barrier disruption via claudin-2 and cellular metabolism via AMPK in airway epithelial Calu-3 cells.

Yuki Kodera, Hirofumi Chiba, Takumi Konno, Takayuki Kohno, Hiroki Takahashi, Takashi Kojima

Biochemical and biophysical research communications 527 ( 2 ) 553 - 560 2020.06 [Refereed] [International journal]

A non-histone chromatin-associated protein, high mobility group box 1 (HMGB1), which impairs the airway epithelial barrier, is involved in the induction of airway inflammation in patients with allergy, asthma, chronic obstructive pulmonary disease (COPD), and idiopathic pulmonary fibrosis (IPF). Tricellular tight junctions (tTJs) form at the convergence of bicellular tight junctions (bTJs). Angulin-1/lipolysis-stimulated lipoprotein receptor (LSR) is a novel molecule present at tricellular contacts and contributes to the epithelial barrier and cellular metabolism. Adenosine monophosphate-activated protein kinase (AMPK) is a central metabolic regulator and has a reciprocal association with TJs. In the present study, to examine how HMGB1 contributes to airway epithelial barrier disruption and the cellular metabolism indicated as mitochondrial respiration, bronchial epithelial Calu-3 cells were transfected with siRNAs of angulin-1/LSR or treated with HMGB1 and the relationship between HMGB1 and angulin-1/LSR was investigated. Knockdown of angulin-1/LSR upregulated the expression of the tight junction molecule claudin-2, AMPK activity, and mitochondrial respiration, and downregulated the epithelial barrier. Treatment with HMGB1 downregulated angulin-1/LSR expression and the epithelial barrier, and upregulated claudin-2 expression, AMPK activity and mitochondrial respiration. Treatment with EW-7197, a transforming growth factor-β (TGF-β) type I receptor kinase inhibitor, prevented all the effects of HMGB1 in Calu-3 cells. HMGB1-downregulated angulin-1/LSR induced epithelial barrier disruption via claudin-2 and cellular metabolism via AMPK in airway epithelial Calu-3 cells. The effects of HMGB1 contribute to TGF-β signaling and EW-7197 shows potential for use in therapy for HMGB1-induced airway inflammation.

DOI PubMed

Glucose-Dependent FOXM1 Promotes Epithelial-to-Mesenchymal Transition Via Cellular Metabolism and Targeting Snail in Human Pancreatic Cancer.

Takuro Kyuno, Takayuki Kohno, Takumi Konno, Hiroshi Yamaguchi, Daisuke Kyuno, Masafumi Imamura, Yasutoshi Kimura, Takashi Kojima, Ichiro Takemasa

Pancreas 49 ( 2 ) 273 - 280 2020.02 [Refereed] [International journal]

OBJECTIVES: Transcription factor Forkhead box protein M1 (FOXM1) plays critical roles in the progression of cancer including epithelial-to-mesenchymal transition (EMT). The aim of this study is to characterize the regulatory mechanisms of FOXM1 in EMT via pancreatic cancer metabolism. METHODS: We investigated the regulation of EMT via mitochondrial respiration by FOXM1 using pancreatic cancer cell lines HPAC and PANC-1 and normal human pancreatic duct epithelial cells. RESULTS: Forkhead box protein M1 and Snail were strongly expressed in HPAC and PANC-1. Epithelial-to-mesenchymal transition-modulated claudin-1 level was lower in PANC-1 than in HPAC. In both cell lines in low-glucose medium, FOXM1 and Snail were decreased and claudin-1 was increased. Knockdown of FOXM1 increased claudin-1 and decreased Snail in both cell lines. Low-glucose medium and downregulation of FOXM1 inhibited the cell migration in both cell lines. In both cell lines, mitochondrial respiration was at higher levels in low-glucose medium than in high-glucose medium. Downregulation of FOXM1 induced mitochondrial respiration in high-glucose medium. In normal human pancreatic duct epithelial cells, FOXM1 and Snail were low and claudin-1 was highly expressed, whereas overexpression of FOXM1 decreased claudin-1. CONCLUSIONS: Glucose-dependent FOXM1 promoted EMT via Snail and pancreatic cancer metabolism.

DOI PubMed

Localization of Tricellular Tight Junction Molecule LSR at Midbody and Centrosome During Cytokinesis in Human Epithelial Cells.

Takumi Konno, Takayuki Kohno, Shin Kikuchi, Hiroshi Shimada, Seiro Satohisa, Kenichi Takano, Tsuyoshi Saito, Takashi Kojima

The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society 68 ( 1 ) 59 - 72 2020.01 [Refereed] [International journal]

Epithelial integrity and barrier function are maintained during cytokinesis in vertebrate epithelial tissues. The changes in localization and the roles of tricellular tight junction molecule lipolysis-stimulated lipoprotein receptor (LSR) during cytokinesis are not well known, although new tricellular tight junctions form at the flank of the midbody during cytokinesis. In this study, we investigated the changes in localization and the role of LSR at the midbody and centrosome during cytokinesis using human endometrial carcinoma cell line Sawano, comparing the tricellular tight junction molecule tricellulin; bicellular tight junction molecules occludin, claudin-7, zonula occludens-1, and cingulin; and the epithelial polarized related molecules apoptosis-stimulating of p53 protein 2, PAR3, and yes-associated protein. During cytokinesis induced by treatment with taxol, the epithelial barrier was maintained and the tricellular tight junction molecules LSR and tricellulin were concentrated at the flank of the acetylated tubulin-positive midbody and in γ-tubulin-positive centrosomes with the dynein adaptor Hook2, whereas the other molecules were localized there as well. All the molecules disappeared by knockdown using small interfering RNAs. Furthermore, by the knockdown of Hook2, the epithelial barrier was maintained and most of the molecules disappeared from the centrosome. These findings suggest that LSR may play crucial roles not only in barrier function but also in cytokinesis.

DOI PubMed

Tricellular tight junction protein LSR/angulin-1 contributes to the epithelial barrier and malignancy in human pancreatic cancer cell line.

Takuro Kyuno, Daisuke Kyuno, Takayuki Kohno, Takumi Konno, Shin Kikuchi, Chihiro Arimoto, Hiroshi Yamaguchi, Masafumi Imamura, Yasutoshi Kimura, Masuo Kondoh, Ichiro Takemasa, Takashi Kojima

Histochemistry and cell biology 153 ( 1 ) 5 - 16 2020.01 [Refereed] [International journal]

Lipolysis-stimulated lipoprotein receptor (LSR)/angulin-1 is a crucial molecule of tricellular contacts in the epithelial barrier of normal cells and the malignancy of cancer cells. To investigate whether LSR/angulin-1 affects the epithelial barrier and malignancy in human pancreatic cancer, human pancreatic cancer cell line HPAC was used. Treatment with EGF or TGF-β increased the expression of LSR, but not tricellulin (TRIC), and induced the localization of LSR and TRIC to bicellular tight junctions from tricellular tight junctions. TGF-β receptor type-1 inhibitor EW-7197 prevented changes of the distribution and the barrier function of LSR by TGF-β. Knockdown of LSR increased cell migration, invasion, proliferation and EGF ligand amphiregulin expression and decreased the epithelial barrier. Treatment with amphiregulin induced cell migration and invasion and knockdown of amphiregulin prevented the increases of cell migration, invasion and proliferation caused by knockdown of LSR. Treatment with LSR ligand peptide angubindin-1 decreased the epithelial barrier and the expression of LSR, but not TRIC, and increased cell invasion. Knockdown of TRIC decreased cell migration and the epithelial barrier. In immunohistochemical analysis of human pancreatic cancer tissues, LSR and TRIC were found to be localized at the cell membranes of normal pancreatic ducts and well-differentiated pancreatic ductal adenocarcinomas (PDAC), whereas in poorly differentiated PDAC, LSR was weakly detected in the cytoplasm. Amphiregulin was highly expressed in the cytoplasm of well- and poorly differentiated PDAC. In pancreatic cancer, LSR contributes to the epithelial barrier and malignancy via growth factors and may be a potential targeting molecule in the therapy.

DOI PubMed

Epithelial barrier dysfunction and cell migration induction via JNK/cofilin/actin by angubindin-1.

Takumi Konno, Takayuki Kohno, Shin Kikuchi, Hiroshi Shimada, Seiro Satohisa, Tsuyoshi Saito, Masuo Kondoh, Takashi Kojima

Tissue barriers 8 ( 1 ) 1695475 - 1695475 2020 [Refereed] [International journal]

Angulin-1/LSR is a tricellular tight junction molecule, that plays an important role in maintaining the epithelial and endothelial barriers. The actin cytoskeleton at tricellular contacts also contributes to the maintenance of the epithelial barrier. Loss of angulin-1/LSR enhances the migration of various cancer cells. Angubindin-1 is a novel binder to angulin-1/LSR and angulin-3. It is a peptide generated from the angulin-1 binding site of Clostridium perfringens iota toxin, which affects the actin cytoskeleton and decreases the epithelial and endothelial barrier functions. However, its regulatory mechanisms are not well understood. To investigate the regulatory mechanisms of the epithelial barrier dysfunction and cell migration induction by angubindin-1, we used human endometrial cancer cell line Sawano, which has high LSR expression and the epithelial barrier function. Angubindin-1 decreased LSR expression and the epithelial barrier function and increased cell migration. It inhibited the recovery of the epithelial barrier function in a Ca-switch model. At tricellular contacts, sinking of the membrane and an increase of actin fibers near the junctions were caused by angubindin-1. It dynamically changed F-actin from lines to dot-like structures at tricellular contacts. Angubindin-1 transiently increased the phosphorylation of cofilin and JNK, which are involved in the regulation of the intracellular actin cytoskeleton. Furthermore, knockdown of JNK and the JNK inhibitor SP600125 prevented the decrease of the epithelial barrier function and the increase of cell migration induced by angubindin-1. These findings suggest that angubindin-1 might reversibly regulate the epithelial barrier and cell migration at tricellular contacts via JNK/cofilin/actin cytoskeleton dynamics.

DOI PubMed

Rho-kinase and PKCα Inhibition Induces Primary Cilia Elongation and Alters the Behavior of Undifferentiated and Differentiated Temperature-sensitive Mouse Cochlear Cells.

Akito Kakiuchi, Takayuki Kohno, Takuya Kakuki, Yakuto Kaneko, Takumi Konno, Yukino Hosaka, Tomohiro Hata, Shin Kikuchi, Takafumi Ninomiya, Tetsuo Himi, Kenichi Takano, Takashi Kojima

The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society 67 ( 7 ) 523 - 535 2019.07 [Refereed] [International journal]

Primary cilia, regulated via distinct signal transduction pathways, play crucial roles in various cellular behaviors. However, the full regulatory mechanism involved in primary cilia development during cellular differentiation is not fully understood, particularly for the sensory hair cells of the mammalian cochlea. In this study, we investigated the effects of the Rho-kinase inhibitor Y27632 and PKCα inhibitor GF109203X on primary cilia-related cell behavior in undifferentiated and differentiated temperature-sensitive mouse cochlear precursor hair cells (the conditionally immortalized US/VOT-E36 cell line). Our results indicate that treatment with Y27632 or GF109203X induced primary cilia elongation and tubulin acetylation in both differentiated and undifferentiated cells. Concomitant with cilia elongation, Y27632 treatment also increased Hook2 and cyclinD1 expression, while only Hook2 expression was increased after treatment with GF109203X. In the undifferentiated cells, we observed an increase in the number of S and G2/M stage cells and a decrease of G1 cells after treatment with Y27632, while the opposite was observed after treatment with GF109203X. Finally, while both treatments decreased oxidative stress, only treatment with Y27632, not GF109203X, induced cell cycle-dependent cell proliferation and cell migration.

DOI PubMed

Induction of airway progenitor cells via p63 and KLF11 by Rho-kinase inhibitor Y27632 in hTERT-human nasal epithelial cells.

Yakuto Kaneko, Takumi Konno, Takayuki Kohno, Takuya Kakuki, Ryo Miyata, Tsuyoshi Ohkuni, Akito Kakiuchi, Ryoto Yajima, Kizuku Ohwada, Makoto Kurose, Tetsuo Himi, Kenichi Takano, Takashi Kojima

American journal of translational research 11 ( 2 ) 599 - 611 2019 [Refereed] [International journal]

Rho-kinase inhibitor Y27632, which is a factor in conditional reprogramming culture, induces airway progenitor clone formation. To investigate whether Y27632 enhances airway progenitor cells in nasal epithelium, primary cultures of HNECs transfected with human telomerase reverse transcriptase (hTERT-HNECs) were treated with Y27632. In TERT-HNECs treated with Y27632 for 5 days, upregulation of p63, gap junction molecules Cx26, Cx30, Cx43, cytochrome P450 enzymes CYP2C9, CYP2C18, CYP39A1, CYP4B1, CYP2G1P, CYP4Z1, and KLF families KLF10 and KLF11 were observed compared to the control. Downregulation of tight junction molecules claudin-4, -7, and -23 was observed. Circumfential submembrane F-actin was also induced. The functions of gap junctional intercellular communication (GJIC) and the epithelial barrier were upregulated. Knockdown of p63 by siRNAs of TAp63 or ΔNp63 inhibited Cx26, Cx43 and CYP2C18, and induced claudin-1, and -4. Knockdown of KLF11 prevented p63 expression and enhancement of the epithelial barrier function by Y27632. In nasal mucosal tissues from patients with allergic rhinitis (AR), localized alteration of p63, KLF11, RhoA, Cx30 and claudin-4 was observed. Treatment with Y27632 in long-term culture induced airway progenitor cells via KLF11 in p63-positive human nasal epithelium. Airway progenitor cells of nasal epithelium induced by Y27632 is important in understanding upper airway disease-specific characteristics.

PubMed

Mechanism of fibrogenesis in submandibular glands in patients with IgG4-RD.

Ryoto Yajima, Kenichi Takano, Takumi Konno, Takayuki Kohno, Yakuto Kaneko, Takuya Kakuki, Kazuaki Nomura, Akito Kakiuchi, Tetsuo Himi, Takashi Kojima

Journal of molecular histology 49 ( 6 ) 577 - 587 2018.12 [Refereed] [International journal]

The aim of this study was to investigate the mechanisms driving fibrosis in the submandibular glands (SMG) of patients with IgG4-related disease (IgG4-RD). Immunohistochemistry showed that many fibroblast-like cells expressing IL-6, IL-18, TSLP, IL-33, and MMP1 were present in SMG from the affected patients. SMG fibroblasts were derived from patients with or without IgG4-RD and were cultured in vitro. Expression of IL-6, IL-18, TSLP, IL-33 and MMP1, the secretion of IL-6 and G2/M phase were upregulated in the fibroblasts from the affected patients. By treatment with inflammatory cytokines IL-1β, TNFα or TGF-β after treatment with or without the NF-κB inhibitor curcumin, curucumin blocked the production and secretion of IL-6 upregulated by IL-1β, TNFα, or TNFα/TGF-β in all fibroblasts. Wnt1-inducible signaling protein 1 (WISP1), which can enhance fibroblasts proliferation, was also more abundantly expressed in affected fibroblasts, while treatment with IL-6 induced WISP1, treatment with WISP1 increased the G2/M phase, and curucumin inhibited WISP1 induced by TNFα/TGF-β in unaffected fibroblasts. IL-33 in affected fibroblasts was induced by IL-1β, TNFα, or TNFα/TGF-β, while the effect of IL-1β or TNFα/TGF-β was blocked by curcumin. These results suggest fibrosis in the SMG of affected patients is closely linked to the proliferation of fibroblasts following induction of IL-6 and WISP1 by inflammatory cytokines. The Th2 cytokines TSLP and IL-33 are also upregulated in affected SMG, and thus may cause chronic inflammation and IgG4 accumulation.

DOI PubMed

Guanylate binding protein-1-mediated epithelial barrier in human salivary gland duct epithelium.

Takumi Konno, Kenichi Takano, Yakuto Kaneko, Takuya Kakuki, Kazuaki Nomura, Ryoto Yajima, Akito Kakiuchi, Takayuki Kohno, Tetsuo Himi, Takashi Kojima

Experimental cell research 371 ( 1 ) 31 - 41 2018.10 [Refereed] [International journal]

Guanylate-binding protein-1 (GBP-1) is an interferon-inducible large GTPase involved in the epithelial barrier at tight junctions. To investigate the role of GBP-1 in the epithelial barrier, primary human salivary gland duct epithelial cells were treated with the the proinflammatory cytokines IFNγ, IL-1β, TNFα and the growth factor TGF-β. Treatment with IFNγ, IL-1β, or TNFα markedly enhanced GBP-1 and the epithelial barrier function, and induced not only CLDN-7 but also the tricellular tight junction molecule lipolysis-stimulated lipoprotein receptor (LSR). Knockdown of GBP-1 by its siRNA induced endocytosis of tight junction molecules, and prevented the increases of CLDN-7 and LSR with the upregulation of the epithelial barrier function induced by treatment with IFNγ or TNFα. Treatment with a PKCα inhibitor induced expression of GBP-1, CLDN-7 and LSR and enhanced the epithelial barrier function. In almost intact salivary gland ducts from patients with IgG4-related disease (IgG4-RD) indicated significant infiltration of IgG-positive plasma cells, expression of GBP-1, CLDN-7 and LSR was increased. These findings indicated that GBP-1 might play a crucial role in barrier function of normal human salivary gland duct epithelium and perform a preventive role in the duct epithelium of IgG4-RD disease.

DOI PubMed

The role of claudin-2 on the malignancy of human endometrioid carcinoma tissues

Satohisa Seiro, Shimada Hiroshi, Kohno Takayuki, Konno Takumi, Tamate Masato, Matsuura Motoki, Teramoto Mizue, Akimoto Taishi, Iwasaki Masahiro, Kojima Takashi, Saito Tsuyoshi

International Journal of Gynecological Cancer ( BMJ PUBLISHING GROUP ) 28 109 - 109 2018.10 [Refereed]

Cobalt inhibits motility of axonal mitochondria and induces axonal degeneration in cultured dorsal root ganglion cells of rat.

Shin Kikuchi, Takafumi Ninomiya, Takayuki Kohno, Takashi Kojima, Haruyuki Tatsumi

Cell biology and toxicology 34 ( 2 ) 93 - 107 2018.04 [Refereed] [International journal]

Cobalt is a trace element that localizes in the human body as cobalamin, also known as vitamin B12. Excessive cobalt exposure induces a peripheral neuropathy, the mechanisms of which are yet to be elucidated. We investigated how cobalt may affect mitochondrial motility in primary cultures of rat dorsal root ganglion (DRG). We observed mitochondrial motility by time-lapse imaging after DsRed2 tagging via lentivirus, mitochondrial structure using transmission electron microscopy (TEM), and axonal swelling using immunocytochemical staining. The concentration of cobaltous ion (Co2+) required to significantly suppress mitochondrial motility is lower than that required to induce axonal swelling following a 24-h treatment. Exposure to relatively low concentrations of Co2+ for 48 h suppressed mitochondrial motility without leading to axonal swelling. TEM images indicated that Co2+ induces mitochondrial destruction. Our results show that destruction of the axonal mitochondria precedes the axonal degeneration induced by Co2+ exposure.

DOI PubMed

The role of transcriptional factors FOXM1/KLF4 in glucose metabolism during EMT of pancreatic cancer

Kyuno, Takuro, Kojima, Takashi, Konno, Takumi, Kohno, Takayuki, Yamaguchi, Hiroshi, Imamura, Masafumi, Kimura, Yasutoshi, Takemasa, Ichiro

Cancer Science 2018

LOSS OF ASPP2 PROMOTES CELL INVASION AND MIGRATION VIA YAP IN HUMAN ENDOMETRIAL CANCER

Shimada, H, Satohisa, S, Konno, T, Matsuura, M, Teramoto, M, Kohno, T, Iwasaki, M, Kojima, T, Saito, T

International Journal of Gynecological Cancer 2018

Downregulation of lipolysis-stimulated lipoprotein receptor promotes cell invasion via claudin-1-mediated matrix metalloproteinases in human endometrial cancer.

Hiroshi Shimada, Seiro Satohisa, Takayuki Kohno, Takumi Konno, Ken-Ichi Takano, Syunta Takahashi, Tsubasa Hatakeyama, Chihiro Arimoto, Tsuyoshi Saito, Takashi Kojima

Oncology letters ( SPANDIDOS PUBL LTD ) 14 ( 6 ) 6776 - 6782 2017.12 [Refereed] [International journal]

Lipolysis-stimulated lipoprotein receptor (LSR) is a novel molecule present at tricellular contacts which recruits tricellulin (TRIC), a molecular component of tricellular tight junctions (tTJs). LSR and TRIC are colocalized with the bicellular tight junction (bTJ) protein claudin (CLDN)-1-based tight junction strands at tricellular corners. Knockdown of LSR in normal epithelial cells affects tTJ formation and the epithelial barrier function. In cancer cells knockdown of LSR has been demonstrated to increase cell invasion. However, the detailed mechanisms of how the downregulation of LSR enhances cell invasion in cancer remain unclear. In the present study, knockdown of LSR by small interfering RNA (siRNA) in Sawano human endometrial adenocarcinoma cells induced cell invasion. In LSR-knockdown Sawano cells, upregulation of CLDN-1 protein, which contributes to the cell invasion via matrix metalloproteinases (MMPs), was observed compared with the control group by western blotting and immunostaining. Knockdown of LSR significantly induced Sp1 transcription factor activity in the CLDN-1 promoter region. In LSR-knockdown Sawano cells, DNA microarray analysis demonstrated that MMP-1, MMP-2 and MMP-10 mRNA levels were increased, and the protein levels of membrane-type 1-MMP, MMP-2, MMP-9 and MMP-10 were shown to be increased on western blots. Knockdown of CLDN-1 with siRNA prevented the upregulation of cell invasion induced by the knockdown of LSR in Sawano cells. On the invasive front of human endometrial carcinoma tissue samples, a decrease in LSR and increase in CLDN-1 protein levels were observed using immunohistochemical methods. In conclusion, the results indicate that the downregulation of LSR promotes cell invasion of human endometrial cancer via CLDN-1 mediation of MMPs. This mechanism is important for studying the association of tTJs with the cellular invasion of cancer.

DOI PubMed

Regulation of claudin-4 via p63 in human epithelial cells.

Takashi Kojima, Takayuki Kohno, Terufumi Kubo, Yakuto Kaneko, Takuya Kakuki, Akito Kakiuchi, Makoto Kurose, Ken-Ichi Takano, Noriko Ogasawara, Kazufumi Obata, Kazuaki Nomura, Ryo Miyata, Takumi Konno, Shingo Ichimiya, Tetsuo Himi

Annals of the New York Academy of Sciences ( WILEY ) 1405 ( 1 ) 25 - 31 2017.10 [Refereed] [International journal]

P63 is a regulator of cell-cell junction complexes in the epidermis. Claudin-4 is regulated via various factors in normal epithelial cells and diseases. We found that claudin-4 was directly regulated via p63 (TAp63 and ΔNp63) in human keratinocytes and nasal epithelial cells. In the epidermis of atopic dermatitis (AD), which contains ΔNp63-deficient keratinocytes, high expression of claudin-4 was observed. In primary keratinocytes, downregulation of ΔNp63 by treatment with short interfering RNA (siRNA)-p63 induced claudin-4 expression. In nasal epithelial cells in the context of rhinitis or nasal polyps, upregulation of TAp63 and downregulation of claudin-4 were observed. In primary nasal epithelial cells transfected with the human telomerase reverse transcriptase gene, knockdown of p63 by siRNAs induced claudin-4 expression. Taken together, these findings indicate that p63 is a negative regulator of claudin-4 expression. Understanding the regulation of claudin-4 via p63 in human epithelial cells may be important for developing therapies for allergies and drug delivery systems.

DOI PubMed

The role of transcriptional factor p63 in regulation of epithelial barrier and ciliogenesis of human nasal epithelial cells.

Yakuto Kaneko, Takayuki Kohno, Takuya Kakuki, Ken-Ichi Takano, Noriko Ogasawara, Ryo Miyata, Shin Kikuchi, Takumi Konno, Tsuyoshi Ohkuni, Ryoto Yajima, Akito Kakiuchi, Shin-Ichi Yokota, Tetsuo Himi, Takashi Kojima

Scientific reports ( NATURE PUBLISHING GROUP ) 7 ( 1 ) 10935 - 10935 2017.09 [Refereed] [International journal]

Disruption of nasal epithelial tight junctions (TJs) and ciliary dysfunction are found in patients with chronic rhinosinusitis (CRS) and nasal polyps (NPs), along with an increase of p63-positive basal cells and histone deacetylase (HDAC) activity. To investigate these mechanisms, primary cultures of HNECs transfected with human telomerase reverse transcriptase (hTERT-HNECs) were transfected with siRNAs of TAp63 and ΔNp63, treated with the NF-kB inhibitor curucumin and inhibitors of HDACs, and infected with respiratory syncytial virus (RSV). In TERT-HNECs, knockdown of p63 by siRNAs of TAp63 and ΔNp63, induced claudin-1 and -4 with Sp1 activity and enhanced barrier and fence functions. The knockdown of p63 enhanced the number of microvilli with the presence of cilia-like structures. Treatment with curcumin and inhibitors of HDACs, or infection with RSV prevented expression of p63 with an increase of claudin-4 and the number of microvilli. The knockdown or downregulation of p63 inhibited phospho-p38MAPK, and the p38MAPK inhibitor downregulated p63 and upregulated the barrier function. Thus, epithelial barrier and ciliogenesis of nasal epithelium are regulated in a p63-negative manner in normal and upper airway diseases. Understanding of the regulation of p63/p38 MAPK/NF-κB may be important in the therapy for airway allergy and its drug delivery system.

DOI PubMed

がん細胞の上皮バリアおよび悪性化における糖代謝の役割

金野匠, 幸野貴之, 及能大輔, 嶋田浩志, 郷久晴朗, 角木拓也, 金子躍人, 高野賢一, 齋藤豪, 小島隆

日本細胞生物学会大会講演要旨集 ( (一社)日本細胞生物学会 ) 69回 95 - 95 2017.05

細胞密度に依存的なLSRの膜での局在変化はミオシン活性と相関する

幸野貴之, 嶋田浩志, 金野匠, 小島隆

日本病理学会会誌 ( (一社)日本病理学会 ) 106 ( 1 ) 356 - 356 2017.03

Loss of tricellular tight junction protein LSR promotes cell invasion and migration via upregulation of TEAD1/AREG in human endometrial cancer

Hiroshi Shimada, Shyuetsu Abe, Takayuki Kohno, Seiro Satohisa, Takumi Konno, Syunta Takahashi, Tsubasa Hatakeyama, Chihiro Arimoto, Takuya Kakuki, Yakuto Kaneko, Ken-ichi Takano, Tsuyoshi Saito, Takashi Kojima

SCIENTIFIC REPORTS ( NATURE PUBLISHING GROUP ) 7 2017.01 [Refereed]

Lipolysis-stimulated lipoprotein receptor (LSR) is a unique molecule of tricellular contacts of normal and cancer cells. We investigated how the loss of LSR induced cell migration, invasion and proliferation in endometrial cancer cell line Sawano. mRNAs of amphiregulin (AREG) and TEA domain family member 1 (TEAD1) were markedly upregulated by siRNA-LSR. In endometrial cancer tissues, downregulation of LSR and upregulation of AREG were observed together with malignancy, and Yes-associated protein (YAP) was present in the nuclei. siRNA-AREG prevented the cell migration and invasion induced by siRNA-LSR, whereas treatment with AREG induced cell migration and invasion. LSR was colocalized with TRIC, angiomotin (AMOT), Merlin and phosphorylated YAP (pYAP). siRNA-LSR increased expression of pYAP and decreased that of AMOT and Merlin. siRNA-YAP prevented expression of the mRNAs of AREG and TEAD1, and the cell migration and invasion induced by siRNA-LSR. Treatment with dobutamine and 2-deoxy-D-glucose and glucose starvation induced the pYAP expression and prevented the cell migration and invasion induced by siRNA-LSR. siRNA-AMOT decreased the Merlin expression and prevented the cell migration and invasion induced by siRNA-LSR. The loss of LSR promoted cell invasion and migration via upregulation of TEAD1/AREG dependent on YAP/pYAP and AMOT/Merlin in human endometrial cancer cells.

DOI PubMed

Histone deacetylase inhibition prevents cell death induced by loss of tricellular tight junction proteins in temperature-sensitive mouse cochlear cells.

Kenichi Takano, Takuya Kakuki, Yakuto Kaneko, Takayuki Kohno, Shin Kikuchi, Tetsuo Himi, Takashi Kojima

PloS one ( PUBLIC LIBRARY SCIENCE ) 12 ( 8 ) e0182291 2017 [Refereed] [International journal]

Tricellular tight junctions (tTJs) are specialized structures that occur where the corners of three cells meet to seal adjacent intercellular space. The molecular components of tTJs include tricellulin (TRIC) and lipolysis-stimulated lipoprotein receptor (LSR) which recruits TRIC, are required for normal hearing. Although loss of TRIC causes hearing loss with degeneration of cochlear cells, the detailed mechanisms remains unclear. In the present study, by using temperature-sensitive mouse cochlear cells, US/VOT-E36 cell line, we investigated the changes of TRIC and LSR during cochlear cell differentiation and the effects of histone deacetylase (HDAC) inhibitors against cell degeneration induced by loss of TRIC and LSR. During cell differentiation induced by the temperature change, expression of TRIC and LSR were clearly induced. Treatment with metformin enhanced expression TRIC and LSR via AMPK during cell differentiation. Loss of TRIC and LSR by the siRNAs induced cell death in differentiated cells. Treatment with HDAC inhibitors trichostatin A and HDAC6 inhibitor prevented the cell death induced by loss of TRIC and LSR. Collectively, these findings suggest that both tTJ proteins TRIC and LSR have crucial roles for the differentiated cochlear cell survival, and that HDAC inhibitors may be potential therapeutic agents to prevent hearing loss.

DOI PubMed

The Behavior and Role of Lipolysis-stimulated Lipoprotein Receptor, a Component of Tricellular Tight Junctions, in Head and Neck Squamous Cell Carcinomas

Kenichi Takano, Takuya Kakuki, Kazufumi Obata, Kazuaki Nomura, Ryo Miyata, Atsushi Kondo, Makoto Kurose, Akito Kakiuchi, Yakuto Kaneko, Takayuki Kohno, Tetsuo Himi, Takashi Kojima

ANTICANCER RESEARCH ( INT INST ANTICANCER RESEARCH ) 36 ( 11 ) 5895 - 5904 2016.11 [Refereed]

Background/Aim: Lipolysis-stimulated lipoprotein receptor (LSR) knockdown has also been reported to increase the motility and invasiveness of certain cancer cells. Here, we describe, for the first time, the behavior and role of LSR in head and neck squamous cell carcinoma (HNSCC) in vivo and in vitro. Materials and Methods: Samples of HNSCC, normal palatine tonsils, the pharynx carcinoma cell line Detroit562 and primary cultured HNSCC were characterized by immunostaining, western blot, real-time polymerase chain reaction (PCR), Matrigel invasion and proliferation assays. Results: Protein and mRNA of LSR were strongly expressed, as well as claudin-1 in HNSCC tissues than in normal tissues, especially in invasive tissues. Knock-down of LSR and claudin-1 (CLDN-1), but not tricellulin (TRIC) by siRNAs, markedly induced invasiveness of Detroit562 cells and primary cultured HNSCC. LSR inhibited the development and progression of HNSCC. Conclusion: LSR is a potential target for new forms of head and neck cancer therapy.

DOI PubMed

Claudin-binder C-CPE mutants enhance permeability of insulin across human nasal epithelial cells

Takashi Kojima, Masuo Kondoh, Takashi Keira, Ken-Ichi Takano, Takuya Kakuki, Yakuto Kaneko, Ryo Miyata, Kazuaki Nomura, Kazufumi Obata, Takayuki Kohno, Takumi Konno, Norimasa Sawada, Tetsuo Himi

Drug Delivery ( Taylor and Francis Ltd ) 23 ( 8 ) 2703 - 2710 2016.10 [Refereed]

Objective: Intranasal insulin administration has therapeutic potential for Alzheimer's disease and in intranasal administration across the nasal mucosa, the paracellular pathway regulated by tight junctions is important. The C-terminal fragment of Clostridium perfringens enterotoxin (C-CPE) binds the tight junction protein claudin and disrupts the tight junctional barrier without a cytotoxic effect. The C-CPE mutant called C-CPE 194 binds only to claudin-4, whereas the C-CPE 194 mutant called C-CPE m19 binds not only to claudin-4 but also to claudin-1. Methods: In the present study, to investigate the effects of C-CPE mutants on the tight junctional functions of human nasal epithelial cells (HNECs) and on the permeability of human recombinant insulin across the cells, HNECs were treated with C-CPE 194 and C-CPE m19. Results: C-CPE 194 and C-CPE m19 disrupted the barrier and fence functions without changes in expression of claudin-1, -4, -7, and occludin or cytotoxicity, whereas they transiently increased the activity of ERK1/2 phosphorylation. The disruption of the barrier function caused by C-CPE 194 and C-CPE m19 was prevented by pretreatment with the MAPKK inhibitor U0126. Furthermore, C-CPE 194 and C-CPE m19 significantly enhanced the permeability of human recombinant insulin across HNECs and the permeability was also inhibited by U0126. Conclusion: These findings suggest that C-CPE mutants 194 and m19 can regulate the permeability of insulin across HNECs via the MAPK pathway and may play a crucial role in therapy for the diseases such as Alzheimer's disease via the direct intranasal insulin administration.

DOI PubMed

Dysregulation of junctional adhesion molecule-A via p63/GATA-3 in head and neck squamous cell carcinoma

Takuya Kakuki, Makoto Kurose, Ken-ichi Takano, Atsushi Kondoh, Kazufumi Obata, Kazuaki Nomura, Ryo Miyata, Yakuto Kaneko, Takumi Konno, Syunta Takahashi, Tsubasa Hatakeyama, Takayuki Kohno, Tetsuo Himi, Takashi Kojima

ONCOTARGET ( IMPACT JOURNALS LLC ) 7 ( 23 ) 33887 - 33900 2016.06 [Refereed]

Junctional adhesion molecule-A (JAM-A), which belongs to the IgG superfamily, is a tight junction molecule associated with epithelial and endothelial barrier function. Overexpression of JAM-A is also closely associated with invasion and metastasis of cancers such as breast cancer, lung cancer and pancreatic cancer. However, little is known about the mechanism in overexpression of JAM-A in head and neck squamous cell carcinoma (HNSCC). In the present study, we found high expression of JAM-A at the protein and mRNA levels in HNSCC tissues, including those of the oropahrynx, larynx, and hypopharynx, together with high protein expression of beta-catenin, p63, Delta Np63 and GATA-3. Furthermore, in ELISA, a significant increase of soluble JAM-A in the sera of HNSCC patients was observed compared to healthy subjects. Knockdown of JAM-A by siRNA inhibited cell proliferation, invasion and migration in the HNSCC cell line Detroit562 in vitro. JAM-A expression in Detroit562 was increased via a distinct signal transduction pathway including NF-kappa B. Expression of JAM-A, beta-catenin, p63 and Delta Np63 in Detroit562 was decreased under hypoxia. Knockdown of p63, Delta Np63 or GATA-3 by siRNAs reduced JAM-A expression in Detroit562. In primary cultured HNSCC cells in which CK7, p63, Delta Np63 and GATA-3 were detected, JAM-A expression was decreased by knockdown of p63 or Delta Np63. These results indicate that JAM-A is a biomarker of malignancy in HNSCC and that plasma soluble JAM-A may contribute to serum-based diagnosis of HNSCC. The mechanism of dysregulation of JAM-A via p63/GATA-3 is important in possible molecular targeted therapy for HNSCC.

DOI PubMed

The roles of tricellular tight junction protein lipolysis-stimulated lipoprotein receptor in malignancy of human endometrial cancer cells

Hiroshi Shimada, Seiro Satohisa, Takayuki Kohno, Syunta Takahashi, Tsubasa Hatakeyama, Takumi Konno, Mitsuhiro Tsujiwaki, Tsuyoshi Saito, Takashi Kojima

ONCOTARGET ( IMPACT JOURNALS LLC ) 7 ( 19 ) 27735 - 27752 2016.05 [Refereed]

Lipolysis-stimulated lipoprotein receptor (LSR) has been identified as a novel molecular constituent of tricellular contacts that have a barrier function for the cellular sheet. LSR recruits tricellulin (TRIC), which is the first molecular component of tricellular tight junctions. Knockdown of LSR increases cell motility and invasion of certain cancer cells. However, the behavior and the roles of LSR in endometrial cancer remain unknown. In the present study, we investigated the behavior and roles of LSR in normal and endometrial cancer cells in vivo and in vitro. In endometriosis and endometrial cancer, LSR was observed not only in the subapical region but also throughout the lateral region as well as in normal endometrial epithelial cells in the secretory phase, and LSR in the cancer was reduced in correlation with the malignancy. Knockdown of LSR by the siRNA in cells of the endometrial cancer cell line Sawano, induced cell migration, invasion and proliferation, while TRIC relocalized from the tricellular region to the bicellular region at the membrane. In Sawano cells and normal HEEs, a decrease of LSR induced by leptin and an increase of LSR induced by adiponectin and the drugs for type 2 diabetes metformin and berberine were observed via distinct signaling pathways including JAK2/STAT. In Sawano cells, metformin and berberine prevented cell migration and invasion induced by downregulation of LSR by the siRNA and leptin treatment. The dissection of the mechanism in the downregulation of endometrial LSR during obesity is important in developing new diagnostic and therapy for endometrial cancer.

DOI PubMed

Behavior of primary cilia and tricellular tight junction proteins during differentiation in temperature-sensitive mouse cochlear precursor hair cells

Takuya Kakuki, Yakuto Kaneko, Kenichi Takano, Takafumi Ninomiya, Takayuki Kohno, Takashi Kojima, Tetsuo Himi

Advances in Oto-Rhino-Laryngology ( S. Karger AG ) 77 27 - 32 2016 [Refereed]

In the sensory hair cells of the mammalian cochlea, the primary cilia in the planar cell polarity as well as the tight junctions in the epithelial cell polarity and the barrier are important to maintain normal hearing. Temperature-sensitive mouse cochlear precursor hair cells were used to investigate the behavior of primary cilia and tricellular tight junction proteins during the differentiation of sensory hair cells. In undifferentiated cells (incubated at 33°C), many acetylated tubulin-positive primary cilia were observed, and each was accompanied with an γ-tubulin-positive basal body. The primary cilia had a '9 + 0' architecture with nine outer microtubule doublets but lacking a central pair of microtubules. In differentiated cells (incubated at 39°C), acetylated tubulin-positive primary cilia as well as acetylated tubulin-positive cilia-like structures were partially observed on the cell surface. In differentiated cells, the number of primary cilia was markedly reduced compared with undifferentiated cells, and innumerable cilia-like structures with no ciliary pockets were partially observed on the cell surface. In undifferentiated cells, few tricellulin molecules and lipolysis-stimulated lipoprotein receptors (LSRs) were observed in the cytoplasm. In differentiated cells, many tricellulin molecules and LSRs were observed on the membranes and within the cytoplasm. Conditional immortalized mouse cochlear precursor hair cells may be useful to investigate the roles of primary cilia and tricellular tight junctions during cellular differentiation and degeneration such as apoptosis.

DOI PubMed

Junctional adhesion molecule - A in head and neck squamous cell carcinoma

Makoto Kurose, Takuya Kakuki, Kenichi Takano, Atsushi Kondo, Kazufumi Obata, Kazuaki Nomura, Ryo Miyata, Yakuto Kaneko, Takumi Konno, Takayuki Kohno, Takashi Kojima, Tetsuo Himi

Advances in Oto-Rhino-Laryngology ( S. Karger AG ) 77 92 - 97 2016 [Refereed]

Junctional adhesion molecule-A (JAM-A), which belongs to the IgG superfamily, is one of the tight junction molecules. JAM-A is dysregulated in various cancers and is closely associated with the invasion and metastasis of cancers such as breast cancer, lung cancer and pancreatic cancer. In the present study, we found a high expression of JAM-A in head and neck squamous cell carcinoma (HNSCC) as well as β-catenin in immunohistochemistry. The expression of JAM-A and β-catenin was of a low level in differentiation-induced cancer pearl regions of HNSCC. Real-time PCR showed the high expression of JAM-A mRNA through all differentiated stages (well, moderate, poor) of HNSCC. When we performed ELISA using the serum of HNSCC patients to measure plasma-soluble JAM-A, it was found to be higher in HNSCC patients than healthy subjects. These results indicate that JAM-A is one of the malignancy markers of HNSCC as well as β-catenin in histopathology, and the plasma-soluble JAM-A may contribute to a serum diagnosis of HNSCC. JAM-A is a promising molecular target for diagnosis and therapy in HNSCC.

DOI PubMed

Claudin-4 binder C-CPE 194 enhances effects of anticancer agents on pancreatic cancer cell lines via a MAPK pathway.

Tsuyoshi Kono, Masuo Kondoh, Daisuke Kyuno, Tatsuya Ito, Yasutoshi Kimura, Masafumi Imamura, Takayuki Kohno, Takumi Konno, Tomohisa Furuhata, Norimasa Sawada, Koichi Hirata, Takashi Kojima

Pharmacology research & perspectives 3 ( 6 ) e00196 2015.12 [Refereed] [International journal]

The C-terminal fragment of Clostridium perfringens enterotoxin (C-CPE) modulates the tight junction protein claudin and disrupts the tight junctional barrier. It also can enhance the effectiveness of anticancer agents. However, the detailed mechanisms of the effects of C-CPE remain unclear in both normal and cancerous cells. The C-CPE mutant called C-CPE 194 binds only to claudin-4, but the C-CPE 194 mutant called C-CPE m19 binds not only to claudin-4 but also to claudin-1. In the present study, to investigate the mechanisms of the effects of C-CPE on claudin expression, the tight junctional functions and the cytotoxicity of anticancer agents, human pancreatic cancer cells, and normal human pancreatic duct epithelial cells (HPDEs) were treated with C-CPE 194 and C-CPE m19. In well-differentiated cells of the pancreatic cancer cell line HPAC, C-CPE 194 and C-CPE m19 disrupted both the barrier and fence functions without changes in expression of claudin-1 and -4, together with an increase of MAPK phosphorylation. C-CPE 194, but not C-CPE m19, enhanced the cytotoxicity of the anticancer agents gemcitabine and S-1. In poorly differentiated pancreatic cancer cell line PANC-1, C-CPE 194, but not C-CPE m19, decreased claudin-4 expression and enhanced MAPK activity and the cytotoxicity of the anticancer agents. In normal HPDEs, C-CPE 194 and C-CPE m19 decreased claudin-4 expression and enhanced the MAPK activity, whereas they did not affect the cytotoxicity of the anticancer agents. Our findings suggest that the claudin-4 binder C-CPE 194 enhances effects of anticancer agents on pancreatic cancer cell lines via a MAPK pathway.

DOI PubMed

肥満によるヒト子宮内膜癌の悪性化におけるlipolysis-stimulated lipoprotein receptorの役割

小島隆, 嶋田浩志, 郷久晴朗, 金野匠, 高橋俊太, 畠山翔翼, 有元千尋, 野島伊世里, 齋藤豪, 幸野貴之

日本生化学会大会・日本分子生物学会年会合同大会講演要旨集 ( (公社)日本生化学会 ) 88回・38回 [3T18p - 01(3P1048)] 2015.12

Poly(I:C) induced microRNA-146a regulates epithelial barrier and secretion of proinflammatory cytokines in human nasal epithelial cells

Ryo Miyata, Takuya Kakuki, Kazuaki Nomura, Tsuyoshi Ohkuni, Noriko Ogasawara, Ken-ichi Takano, Takumi Konno, Takayuki Kohno, Norimasa Sawada, Tetsuo Himi, Takashi Kojima

EUROPEAN JOURNAL OF PHARMACOLOGY ( ELSEVIER SCIENCE BV ) 761 375 - 382 2015.08 [Refereed]

Human nasal epithelial cells (HNECs) are important in the tight junctional barrier and innate immune defense protecting against pathogens invading via Toll-like receptors (TERs). MicroRNAs (miRNAs) I egulate expression of tight junctions as direct or indirect targeting genes and maintain the barrier Function. However, the roles of miRNAs in the epithelial barrier of HNECs via TLRs remain unknown. In the present study, to investigate the effects of miRNAs on the epithelial barrier of HNECs via TLRs, primary cultured HNECs transfected with human telomerase reverse transcriptase (hTERT-HNECs), were treated with the TLR3 ligand poly(I:C) and miRNA array analysis was performed. In the miRNA array of the cells treated with poly(I:C), upregulation of miR-187, -146a, -574, -4274, -4433, -4455 and -4750, and downregulation of miR-4785 by more than twofold compared to the control were observed. When control HNECs were treated with mimics and inhibitors of these miRNAs, an miR-146a mimic induced expression of tight junction proteins claudin-1, occludin and JAM-A together with an increase of the epithelial barrier function. The poly(I:C)-induced miR-146a was regulated via the distinct TLR3-mediatecl signal pathways PI3K, JNK and NE-kappa B. Furthermore, the miR-146a mimic prevented downregulation of claudin-1 and JAM-A and the secretion of proinflammatory cytokines IL-8 and TNF-alpha induced by poly (I:C) by targeting TRAFG. These findings indicate that, in HNECs, miRNA-146a plays crucial roles in maintenance of the tight junction barrier and innate immune defense protecting against invading pathogens. (C) 2015 Elsevier B.V. All rights reserved.

DOI PubMed

ヒト子宮内膜癌細胞の悪性化におけるlipolysis-stimulated lipoprotein receptorの役割

嶋田浩志, 郷久晴朗, 金野匠, 高橋俊太, 畠山翔翼, 齋藤豪, 幸野貴之, 小島隆

日本細胞生物学会大会講演要旨集 ( (一社)日本細胞生物学会 ) 67回 167 - 167 2015.06

子宮体がん基礎肥満による子宮内膜癌の悪性化におけるLSR(lipolysis-stimulated lipoprotein receptor)の役割

嶋田浩志, 小島隆, 郷久晴朗, 金野匠, 高橋俊太, 畠山翔翼, 幸野貴之, 寺本瑞絵, 田中綾一, 岩崎雅宏, 斎藤豪

日本婦人科腫瘍学会雑誌 ( (公社)日本婦人科腫瘍学会 ) 33 ( 3 ) 535 - 535 2015.06

c-Jun N-terminal kinase inhibitor SP600125 enhances barrier function and elongation of human pancreatic cancer cell line HPAC in a Ca-switch model

Takumi Konno, Takafumi Ninomiya, Takayuki Kohno, Shin Kikuchi, Norimasa Sawada, Takashi Kojima

HISTOCHEMISTRY AND CELL BIOLOGY ( SPRINGER ) 143 ( 5 ) 471 - 479 2015.05 [Refereed]

c-Jun N-terminal kinase (JNK), known as a stress-activated protein kinase, regulates normal epithelial biological processes, including assembly of adherens and tight junctions, and it is involved in the development of several cancers. The JNK inhibitor SP600125 enhances epithelial barrier function through modulation of tight junction molecules in normal human pancreatic epithelial cells. Furthermore, this JNK inhibitor suppresses the growth of human pancreatic cancer cells. However, the effects of SP600125 on the epithelial barrier in human pancreatic cancer cells remain unknown. In the present study, the JNK inhibitor SP600125 markedly enhanced the barrier function and cell elongation of well-differentiated human pancreatic cancer cell line HPAC in a Ca-switch model. The epithelial barrier function induced by SP600125 was regulated by phosphorylated beta-catenin without changes in the tight junction molecules. The cell elongation induced by SP600125 was closely related to the expression of the F-actin-binding protein DrebrinE. These findings suggest that JNK is involved in the regulation of the epithelial barrier function and cell shape during remodeling of pancreatic cancer cells. The JNK inhibitor SP600125 may have potential as a therapeutic drug for pancreatic cancer via induction of differentiation.

DOI PubMed

Irsogladine Maleate Regulates Gap Junctional Intercellular Communication-Dependent Epithelial Barrier in Human Nasal Epithelial Cells

Ryo Miyata, Kazuaki Nomura, Takuya Kakuki, Ken-ichi Takano, Takayuki Kohno, Takumi Konno, Norimasa Sawada, Tetsuo Himi, Takashi Kojima

JOURNAL OF MEMBRANE BIOLOGY ( SPRINGER ) 248 ( 2 ) 327 - 336 2015.04 [Refereed]

The airway epithelium of the human nasal mucosa acts as the first physical barrier that protects against inhaled substances and pathogens. Irsogladine maleate (IM) is an enhancer of gastric mucosal protective factors via upregulation of gap junctional intercellular communication (GJIC). GJIC is thought to participate in the formation of functional tight junctions. However, the effects of IM on GJIC and the epithelial barrier in human nasal epithelial cells (HNECs) remain unknown. To investigate the effects of IM on GJIC and the tight junctional barrier in HNECs, primary cultures of HNECs transfected with human telomerase reverse transcriptase (hTERT-HNECs) were treated with IM and the GJIC inhibitors oleamide and 18 beta-GA. Some cells were pretreated with IM before treatment with TLR3 ligand poly(I:C) to examine whether IM prevented the changes via TLR3-mediated signal pathways. In hTERT-HNECs, GJIC blockers reduced the expression of tight junction molecules claudin-1, -4, -7, occludin, tricellulin, and JAM-A. IM induced GJIC activity and enhanced the expression of claudin-1, -4, and JAM-A at the protein and mRNA levels with an increase of barrier function. GJIC blockers prevented the increase of the tight junction proteins induced by IM. Furthermore, IM prevented the reduction of JAM-A but not induction of IL-8 and TNF-alpha induced by poly(I:C). In conclusion, IM can maintain the GJIC-dependent tight junctional barrier via regulation of GJIC in upper airway nasal epithelium. Therefore, it is possible that IM may be useful as a nasal spray to prevent the disruption of the epithelial barrier by viral infections and exposure to allergens in human nasal mucosa.

DOI PubMed

Pseudomonas aeruginosa elastase causes transient disruption of tight junctions and downregulation of PAR-2 in human nasal epithelial cells

Kazuaki Nomura, Kazufumi Obata, Takashi Keira, Ryo Miyata, Satoshi Hirakawa, Ken-ichi Takano, Takayuki Kohno, Norimasa Sawada, Tetsuo Himi, Takashi Kojima

RESPIRATORY RESEARCH ( BIOMED CENTRAL LTD ) 15 2014.02 [Refereed]

Background: Pseudomonas aeruginosa causes chronic respiratory disease, and the elastase enzyme that it produces increases the permeability of airway epithelial cells owing to the disruption of tight junctions. P. aeruginosa is also implicated in prolonged chronic rhinosinusitis. However, the effects of P. aeruginosa elastase (PE) against the barrier formed by human nasal epithelial cells (HNECs) remain unknown. Methods: To investigate the mechanisms involved in the disruption of tight junctions by PE in HNECs, primary cultures of HNECs transfected with human telomerase reverse transcriptase (hTERT-HNECs) were used. The hTERT-HNECs were pretreated with inhibitors of various signal transduction pathways, PKC, MAPK, p38MAPK, PI3K, JNK, NF-kappa B, EGF receptor, proteasome, COX1 and COX2 before treatment with PE. Some cells were pretreated with siRNA and agonist of protease activated receptor-2 (PAR-2) before treatment with PE. Expression and structures of tight junctions were determined by Western blotting, real-time PCR, immunostaining and freeze-fracture. Transepithelial electrical resistance (TER) was examined as the epithelial barrier function. Results: PE treatment transiently disrupted the epithelial barrier and downregulated the transmembrane proteins claudin-1 and -4, occludin, and tricellulin, but not the scaffold PDZ-expression proteins ZO-1 and -2 and adherens junction proteins E-cadherin and beta-catenin. The transient downregulation of tight junction proteins was controlled via distinct signal transduction pathways such as the PKC, MAPK, PI3K, p38 MAPK, JNK, COX-1 and -2, and NF-kappa B pathways. Furthermore, treatment with PE transiently decreased PAR-2 expression, which also regulated the expression of the tight junction proteins. Treatment with a PAR-2 agonist prevented the downregulation of the tight junction proteins after PE treatment in HNECs. Conclusions: PE transiently disrupts tight junctions in HNECs and downregulates PAR-2. The transient disruption of tight junctions by PE might occur repeatedly during chronic rhinosinusitis.

DOI PubMed

Expression of tricellulin in epithelial cells and non-epithelial cells

Takashi Kojima, Takafumi Ninomiya, Takumi Konno, Takayuki Kohno, Masahiko Taniguchi, Norimasa Sawada

HISTOLOGY AND HISTOPATHOLOGY ( F HERNANDEZ ) 28 ( 11 ) 1383 - 1392 2013.11 [Refereed]

Tricellulin is the first molecular component of tricellular tight junctions at tricellular contacts where three epithelial cells meet, and it is required for the their formation and maintenance of the epithelial barrier. Tricellulin binds other tight junction proteins, and its expression and distribution are affected by the bicellular tight junction protein occludin and lipolysis-stimulated lipoprotein receptor (LSR) which is expressed at tricellular contacts. Tricellulin is also detected in endothelial cells, neurons, microglia and astrocytes. Here, we focused tricellulin expression in various types of epithelial cells, nasal epithelial cells, pancreatic duct epithelial cells cells and hepatocytes, and non-epithelial cells, dendritic cells and Schwann cells, compared to expression of the bicellular tight junction protein occludin and LSR, and discuss the regulation and the role of tricellulin in cellular specificity.

DOI DOI2 PubMed

FLT3/ITD regulates leukaemia cell adhesion through alpha 4 beta 1 integrin and Pyk2 signalling

Akira Katsumi, Hitoshi Kiyoi, Akihiro Abe, Ryohei Tanizaki, Toshihiro Iwasaki, Miki Kobayashi, Tadashi Matsushita, Kozo Kaibuchi, Takeshi Senga, Tetsuhito Kojima, Takayuki Kohno, Michinari Hamaguchi, Tomoki Naoe

EUROPEAN JOURNAL OF HAEMATOLOGY ( WILEY-BLACKWELL ) 86 ( 3 ) 191 - 198 2011.03 [Refereed]

Internal tandem duplication of FMS-like receptor tyrosine kinase 3 (FLT3/ITD) within its juxtamembrane domain is a frequent mutation in adult acute myeloid leukaemia (AML). This mutation causes constitutive activation of FLT3 and is associated with poor prognosis. The high relapse rate of FLT3/ITD-positive AML might be partly because of insufficient eradication of slow-cycling leukaemic stem cells in the bone marrow microenvironment. beta 1 integrin mediates haematopoietic stem and progenitor cell homing along with their retention in the bone marrow and also inhibits haematopoietic proliferation and differentiation. Here, we demonstrate that inhibition of FLT3/ITD kinase activity by a FLT3 selective inhibitor named FI-700 decreases affinity of alpha 4 beta 1 integrin to soluble VCAM-1. alpha 4 beta 1 integrin deactivation by FI-700 is independent of Rap1, which is the critical regulator of integrin inside-out signalling. In addition, selective inhibition of FLT3/ITD induces Pyk2 dephosphorylation together with the inhibition of phosphatidylinositol-3-kinase (PI3K)/Akt pathway. Both wild-type and ITD-FLT3 proteins co-immunoprecipitated with beta 1 integrin and Pyk2 indicating the signal crosstalk between FLT3, beta 1 integrin and Pyk2. These results collectively indicated that the inhibition of FLT3 kinase might contribute not only to the induction of apoptosis, but also to the leukaemia cell detachment from the bone marrow microenvironment in the treatment of AML.

DOI PubMed

Sphingosine 1-phosphate induces cyclooxygeriase-2 via Ca2+-dependent, but MAPK-independent mechanism in rat vascular smooth muscle cells

Akiko Nodai, Takuji Machida, Sachiko Izumi, Yumika Hamaya, Takayuki Kohno, Yasuyuki Igarashi, Kenji Iizuka, Masaru Minami, Masahiko Hirafuji

LIFE SCIENCES ( PERGAMON-ELSEVIER SCIENCE LTD ) 80 ( 19 ) 1768 - 1776 2007.04 [Refereed]

The effects of sphingosine 1-phosphate (S1P) on prostaglandin I-2 (PGI(2)) production and cyclooxygenase (COX) expression in cultured rat vascular smooth muscle cells (VSMCs) were investigated. S1P stimulated PGI(2) production in a concentration-dependent manner, which was completely suppressed by NS-398, a selective COX-2 inhibitor, as determined by radioimmunoassay. S1P stimulated COX-2 protein and mRNA expressions in a concentration- and time-dependent manner, while it had no effect on COX-1 expression. S1P(2) and S1P(3) receptors mRNA were abundantly expressed in rat VSMCs. Suramin, an antagonist of S1P(3) receptor, almost completely inhibited S1P-induced COX-2 expression. Pretreatment of VSMCs with pertussis toxin (PTX)partially, but significantly inhibited S1P-inducedPGI(2) production and COX-2 expression. SIP also activated extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK). However, neither PD 98059, a selective inbibitor of ERK activation, nor SB 203580, a selective inhibitor of p38 MAPK, had a significant inhibitory effect on S1P-induced COX-2 expression, suggesting that the MAPK activation does not play main roles in S1P-induced COX-2 induction. S I --P-induced COX-2 expression was inhibited by PP2, an inhibitor of Src-family tyrosine kinase, Ca2+ depletion, and GF 109203X, an inhibitor of protein kinase C (PKC). These results suggest that S 1 P stimulates COX-2 induction in rat VSMCs through mechanisms involving Ca2+-dependent PKC and Src-family tyrosine kinase activation via S I --P-3 receptor coupled to PTX-sensitive and -insensitive G proteins. (C) 2007 Elsevier Inc. All rights reserved.

DOI PubMed

Involvement of Sphingosine 1-Phosphate (S1P) Receptor Type 1 and Type 4 in Migratory Response of Mouse T Cells toward S1P

Hirofumi Matsuyuki, Yasuhiro Maeda, Kazuhiro Yano, Kunio Sugahara, Kenji Chiba, Takayuki Kohno, Yasuyuki Igarashi

CELLULAR & MOLECULAR IMMUNOLOGY ( CHIN SOCIETY IMMUNOLOGY ) 3 ( 6 ) 429 - 437 2006.12 [Refereed]

Sphingosine 1-phosphate (S1P), a pleiotropic lysophospholipid, regulates signal transduction pathway via G-protein-coupled receptors termed S1P(1-5) in several types of the cells including lymphocytes. Higher levels of S1P4 mRNA as well as S1P1 mRNA are expressed in lymphoid tissues such as the spleen, thymus, lymph nodes, and Payer's patches. In contrast to S1P1 that plays an essential role in lymphocyte egress, little is known about the role of S1P4 in immune system. In this study, we found that S1P at 10 to 100 nM significantly induced the cell migration and the significant levels of S1P1 and S1P4 mRNA were expressed in mouse CD4 T cells, D10.G4.1 mouse Th2 cells, and EL-4.IL-2 mouse thymoma cells. In D10.G4.1 and EL-4.IL-2 cells, S1P-induced migration was almost completely inhibited by pretreatment with pertussis toxin, Clostoridium difficile toxin B, and ( S)-enantiomer of FTY720-phosphate, a potent agonist at S1P1 and S1P4. The members of the Rho family small GTPase, Cdc42 and Rac were activated by S1P stimulation in these cells. The transfection with dominant negative or constitutively active forms of Cdc42 and Rac revealed that the activation of both Cdc42 and Rac is essential for S1P-induced migration of these cells. The immunoprecipitation assays using CHO cells co-expressing both S1P4 and S1P1 receptors indicated that S1P4 and S1P1 are associated on the cell surface. These results suggest that the association of S1P4 and S1P1 plays an important role in migratory response of mouse T cells toward S1P.

PubMed

Purification, characterization, cDNA cloning, and expression of asialofetuin-binding C-type lectin from eggs of shishamo smelt (Osmerus [Spirinchus] lanceolatus)

M Hosono, S Sugawara, Y Ogawa, T Kohno, M Takayanagi, K Nitta

BIOCHIMICA ET BIOPHYSICA ACTA-GENERAL SUBJECTS ( ELSEVIER SCIENCE BV ) 1725 ( 2 ) 160 - 173 2005.09 [Refereed]

A novel C-type lectin (OLABL) was isolated from the eggs of shishamo smelt [Osmerus (Spirinchus) lanceolatus] by affinity chromatography on asialofetuin-Sepharose. OLABL had a molecular mass of 29 kDa on SDS-PAGE under nonreducing conditions and two subunits with masses of 15 kDa (OLABL-H) and 14 kDa (OLABL-L) under reducing conditions. Thus, OLABL is a heterodimeric protein. cDNA sequence analysis revealed that the H- and L-subunits of OLABL were composed of 137 and 136 amino acid residues, respectively, and showed almost identical (95%) sequences, with slight differences in the N-terminal and C-terminal regions. Since each subunit contained only the characteristic motif of C-type lectin-like domain (CTLD), EPN-E-WND, OLABL is a member of group VII of the CTLD-containing protein family. Although OLABL had an EPN sequence that is known as a mannosespecific motif found in the collectin family, OLABL agglutinated rabbit erythrocytes without the addition of Ca2+ ion, and this activity was inhibited by L-rhammose and D-galactose derivatives, but not by D-mannose and D-glucose. These results indicate that OLABL has similar characteristics to AJL-2, a calcium-independent lactose specific lectin isolated from Japanese eel skin mucus. Recombinant OLABLs (rHisOLABLs), His-tagged homodimers of the H- and L-subunits, were refolded from inclusion bodies expressed by Escherichia coli. rHisOLABL-L was recovered as a soluble form, but rHisOLABL-H was hardly dissolved in a renaturing buffer. The specific activities of rHisOLABL-L, rHisOLABL-H, and native OLABL were 500, 36, and 20, respectively. These findings suggest that the combination of subunits may affect the solubility and activity of these dimeric form lectins. (c) 2005 Elsevier B.V. All rights reserved.

DOI PubMed

Sphingosine 1-phosphate analogue recognition and selectivity at S1P(4) within the endothelial differentiation gene family of receptors

Y Inagaki, TT Pham, Y Fujiwara, T Kohno, DA Osborne, Y Igarashi, G Tigyi, AL Parrill

BIOCHEMICAL JOURNAL ( PORTLAND PRESS LTD ) 389 187 - 195 2005.07 [Refereed]

Synergistic computational and experimental studies provided previously unforeseen details concerning the structural basis of S1P (sphingosine 1-phosphate) recognition by the S1P(4) G-protein-coupled receptor. Similarly to reports on the S1P(1) receptor, cationic and anionic residues in the third transmembrane domain (R3.28 and E3.29 at positions 124 and 125) form ion pairs with the phosphate and ammonium of S1P, and alanine mutations at these positions abolished specific S1P binding, S1P-induced receptor activation and cell migration. Unlike findings on the S1P(1) receptor, no cationic residue in the seventh transmembrane domain interacts with the phosphate. Additionally, two previously undiscovered interactions with the S1P polar headgroup have been identified. Trp(186) at position 4.64 in the fourth transmembrane domain interacts by a cation-pi interaction with the ammonium group of S1P. Lys(204) at position 5.38 forms an ion pair with the S1P. The S1P(4) and S1P(1) receptors show differences in binding-pocket shape and electrostatic distributions that correlate with the published structure-activity relationships. In particular, the binding pocket of mS1P(4) (mouse S1P(4)) has recognition sites for the anionic phosphate and cationic ammonium groups that are equidistant from the end of the non-polar tail. In contrast, the binding pocket of hS1P(1) (human S1P(4)) places the ammonium recognition site 2 angstrom (1 angstrom = 0.1 nm) closer to the end of the non-polar tail than the phosphate recognition site.

DOI PubMed

Down-regulation of EDG5/S1P2 during myogenic differentiation results in the specific uncoupling of sphingosine 1-phosphate signalling to phospholipase D

E Meacci, F Cencetti, C Donati, F Nuti, M Farnararo, T Kohno, Y Igarashi, P Bruni

BIOCHIMICA ET BIOPHYSICA ACTA-MOLECULAR AND CELL BIOLOGY OF LIPIDS ( ELSEVIER SCIENCE BV ) 1633 ( 3 ) 133 - 142 2003.09 [Refereed]

The bioactive lipid sphingosine 1-phosphate (S1P) is known to exert powerful biological effects through the interaction with various members of the endothelial differentiation gene (EDG) receptor family, recently renamed S1P receptors. In the present study, evidence is provided that differentiation of C2C12 myoblasts into myotubes was accompanied by profound changes of EDG/S1P receptor expression. Indeed, in differentiated cells a significant increase of EDG3/S1P3 together with a large decrease of EDG5/S1P2 expression at mRNA as well as protein level was detected. Moreover, SIP was capable to initiate the signalling pathways downstream to cytosolic Ca2+ increase in myombes, similarly to that observed in myoblasts, whereas the signalling of the bioactive lipid to phospholipase D (PLD), but not that of bradykinin (BK) or lysophosphatidic acid (LPA), was found impaired in differentiated cells. Intriguingly, overexpression of EDG5/S1P2, but not EDG1/S1P1 or EDG3/S1P3, potentiated the efficacy of S1P to stimulate PLD, strongly suggesting a role for EDG5/S1P2 in the signalling to PLD. This view was also supported by the marked reduction of S1P-induced PLD activity in myoblasts loaded with antisense oligodeoxyribonucleotides (ODN) to EDG5/S1P2. Furthermore, overexpression of EDG5/S1P2 rescued the coupling of S1P signalling to PLD in C2C12 myotubes. Experimental evidence here provided supports the notion that EDG5/S1P2 plays a dominant role in the coupling of S1P to PLD in myoblasts and that the down-regulation of the receptor subtype is responsible for the specific uncoupling of S1P signalling to PLD in myotubes. (C) 2003 Elsevier B.V All rights reserved.

DOI PubMed

Synthesis of fluorescence-labeled sphingosine and sphingosine 1-phosphate; Effective tools for sphingosine and sphingosine 1-phosphate behavior

T Hakogi, T Shigenari, S Katsumura, T Sano, T Kohno, Y Igarashi

BIOORGANIC & MEDICINAL CHEMISTRY LETTERS ( PERGAMON-ELSEVIER SCIENCE LTD ) 13 ( 4 ) 661 - 664 2003.02 [Refereed]

A fluorescence-labeled sphingosine and sphingosine 1-phosphate have been successfully synthesized from the oxazolidinone methyl ester derived from glycidol via monoalkylation and the stereoselective reduction of the resulting ketone. The labeled sphingosine was converted into its phosphate by treatment with sphingosine kinase I (SPHK I) from mouse. and in platelets. and it was incorporated into the Chinese Hamster Ovarian (CHO) cells. In addition, MAPK was activated by NBD-Sph-1-P through Edg-1, Sph-1-P receptor. (C) 2003 Elsevier Science Ltd. All rights reserved.

DOI PubMed

Sphingosine 1-phosphate inhibits interleukin-1 beta induction of nitric oxide synthase via extracellular-signal regulated kinase inhibition and Rho in vascular smooth muscle cells.

Y Matsumoto, M Hirafuji, T Machida, S Izumi, A Nodai, T Kohno, Y Igarashi, M Minami

JOURNAL OF PHARMACOLOGICAL SCIENCES ( JAPANESE PHARMACOLOGICAL SOC ) 91 114P - 114P 2003 [Refereed]

Modulation of sphingosine 1-phosphate, a new lipid mediator, on nitric oxide production by vascular smooth muscle cells

Masahiko Hirafuji, Yukihiro Hamaya, Yumika Matsumoto, Takuji Machida, Masaru Minami, Takayuki Kohno, Yasuyuki Igarashi

Folia Pharmacologica Japonica 120 ( 1 ) 2002 [Refereed]

The effects of sphingosine 1-phosphate (SIP) on nitric oxide (NO) production induced by interleukin (IL)-1β in cultured rat vascular smooth muscle cells (VSMCs) have been investigated. Rat VSMCs abundantly expressed mRNA of Edg-3 and Edg-5 receptor subtypes in both unstimulated and IL-1β-stimulated cells. S1P at higher than 0.1 μM inhibited the NO production induced by IL-1β in a concentration-dependent manner, in which S1P at 10 μM caused over 90% inhibition. S1P also inhibited inducible NO synthase (iNOS) protein and mRNA expressions induced by IL-1β in a concentration-dependent manner, which effects were smaller than that in the NO production. S1P also significantly inhibited GTP cyclohydrolase I (GTPCH) mRNA expression induced by IL-1β. PTX pretreatment partially prevented the inhibitory effects of S1P on the NO production and iNOS induction, while completely prevented the inhibitory effects on iNOS and GTPCH mRNA expressions. In PTX-pretreated VSMC, S1P showed a bell-shaped enhancing and inhibitory effect on iNOS protein expression. These results suggest that SIP modulates IL-1β induction of NO production by rat VSMCs through multiple mechanisms, partially via Edg-3 and Edg-5 receptor subtypes coupled to PTX-sensitive G proteins.

PubMed

Apsaragine-linked glycosylation of Edg1, a receptor for sphingosine 1-phosphate: Significance in receptor internalization and receptor-coupled signaling.

Takayuki Kohno, Atsushi Wada, Yasuyuki Igarashi

Chem. Phys. Lipid. 107,39 2000 [Refereed]

Authorship： Lead author

　 PREV - NEXT 　

<KOUNO Takayuki>

Papers - KOUNO Takayuki