Details of a Researcher - KOJIMA Takashi

Papers - KOJIMA Takashi

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Delta Np63 Controls a TLR3-Mediated Mechanism That Abundantly Provides Thymic Stromal Lymphopoietin in Atopic Dermatitis

Terufumi Kubo, Ryuta Kamekura, Ayako Kumagai, Koji Kawata, Keiji Yamashita, Yukari Mitsuhashi, Takashi Kojima, Kotaro Sugimoto, Akihiro Yoneta, Yasuyuki Sumikawa, Toshiharu Yamashita, Noriyuki Sato, Tetsuo Himi, Shingo Ichimiya

PLOS ONE ( PUBLIC LIBRARY SCIENCE ) 9 ( 8 ) e105498 2014.08 [Refereed]

In the skin lesions of atopic dermatitis (AD), keratinocytes release large quantities of thymic stromal lymphopoietin (TSLP), causing unfavorable inflammation along with skin damage. Nevertheless, how TSLP influences keratinocytes themselves is still unknown. In this study, we showed that Delta Np63, a p53-homologue, predominantly expressed in keratinocytes regulated the receptor complex of TSLP, which determines susceptibility to self-derived TSLP. Expression of TSLP receptors in skin tissues and keratinocytes was assessed by immunohistochemistry and quantitative RT-PCR, and in vitro studies were also performed to examine the functional relevance of Delta Np63 in the expression of TSLP receptors and the constituting autocrine and/or paracrine pathway of TSLP under the condition of stimuli to innate receptors sensing cell damage. The results showed that normal keratinocytes in the upper epidermis preferentially expressed TSLP receptors and conversely lacked Delta Np63, which has an inhibitory effect on the expression of TSLP receptors. Interestingly, the epidermis of AD lesions was found to abundantly contain keratinocytes with low or undetectable levels of Delta Np63 (Delta Np63(lo/-)). Moreover, in the absence of Delta Np63, keratinocytes readily presented TSLP and other cytokines by stimuli through Toll-like receptor 3 (TLR3). Together with the evidence that extrinsic TSLP itself augments TSLP production by keratinocytes without Delta Np63, the results indicate that Delta Np63(lo/-) keratinocytes generate TSLP through a putative autocrine and/or paracrine pathway upon TLR3 stimulation within AD lesions, since moieties of damaged cells and pathogens stimulate TLR3.

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

Changes of Tight Junction Protein Claudins in Small Intestine and Kidney Tissues of Mice Fed a DDC Diet.

Abiko Y, Kojima T, Murata M, Tsujiwaki M, Takeuchi M, Sawada N, Mori M

Journal of toxicologic pathology ( 4 ) 26 433 - 438 2013.12 [Refereed]

DOI PubMed

Humulone suppresses replication of respiratory syncytial virus and release of IL-8 and RANTES in normal human nasal epithelial cells

Jun Fuchimoto, Takashi Kojima, Tamaki Okabayashi, Tomoyuki Masaki, Noriko Ogasawara, Kazufumi Obata, Kazuaki Nomura, Satoshi Hirakawa, Naoyuki Kobayashi, Tatsuro Shigyo, Shin-ichi Yokota, Nobuhiro Fujii, Hiroyuki Tsutsumi, Tetsuo Himi, Norimasa Sawada

MEDICAL MOLECULAR MORPHOLOGY ( SPRINGER JAPAN KK ) 46 ( 4 ) 203 - 209 2013.12 [Refereed]

Respiratory syncytial virus (RSV) is the major infectious agent causing serious respiratory tract inflammation in infants and young children. However, an effective vaccine and anti-viral therapy for RSV infection have not yet been developed. Hop-derived bitter acids have potent pharmacological effects on inflammation. Therefore, we investigated the effects of humulone, which is the main constituent of hop bitter acids, on the replication of RSV and release of the proinflammatory cytokine IL-8 and chemokine RANTES in RSV-infected human nasal epithelial cells (HNECs). We found that humulone prevented the expression of RSV/G-protein, formation of virus filaments and release of IL-8 and RANTES in a dose-dependent manner in RSV-infected HNECs. These findings suggest that humulone has protective effects against the replication of RSV, the virus assembly and the inflammatory responses in HNECs and that it is a useful biological product for the prevention and therapy for RSV infection.

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Regulation of tight junctions by sex hormones in normal human endometrial epithelial cells and uterus cancer cell line Sawano.

Someya M, Kojima T, Ogawa M, Ninomiya T, Nomura K, Takasawa A, Murata M, Tanaka S, Saito T, Sawada N

Cell and tissue research ( 2 ) 354 481 - 494 2013.11 [Refereed]

DOI PubMed

Tight junctions in human pancreatic duct epithelial cells.

Takashi Kojima, Hiroshi Yamaguchi, Tatsuya Ito, Daisuke Kyuno, Tsuyoshi Kono, Takumi Konno, Norimasa Sawada

Tissue barriers 1 ( 4 ) e24894 2013.10 [Refereed] [International journal]

Tight junctions of the pancreatic duct are essential regulators of physiologic secretion of the pancreas and disruption of the pancreatic ductal barrier is known to contribute to the pathogenesis of pancreatitis and progression of pancreatic cancer. Various inflammatory mediators and carcinogens can trigger tight junction disassembly and disruption of the pancreatic barrier, however signaling events that mediates such barrier dysfunctions remain poorly understood. This review focuses on structure and regulation of tight junctions in normal pancreatic epithelial cells and mechanisms of junctional disruption during pancreatic inflammation and cancer. We will pay special attention to a novel model of human telomerase reverse transcriptase-transfected human pancreatic ductal epithelial cells and will describe the roles of major signaling molecules such as protein kinase C and c-Jun N-terminal kinase in formation and disassembly of the pancreatic ductal barrier.

DOI PubMed

Protein kinase Cα inhibitor protects against downregulation of claudin-1 during epithelial-mesenchymal transition of pancreatic cancer.

Daisuke Kyuno, Takashi Kojima, Hiroshi Yamaguchi, Tatsuya Ito, Yasutoshi Kimura, Masafumi Imamura, Akira Takasawa, Masaki Murata, Satoshi Tanaka, Koichi Hirata, Norimasa Sawada

Carcinogenesis 34 ( 6 ) 1232 - 43 2013.06 [Refereed] [International journal]

Protein kinase Cα (PKCα) is highly expressed in pancreatic cancer. However, the effects of PKCα on Snail and claudin-1, which play crucial roles in epithelial cell polarity during epithelial-mesenchymal transition (EMT), remain unclear. In this study, we investigated the mechanisms of regulation of Snail and claudin-1 via a PKCα signal pathway during EMT in pancreatic cancer cells and in normal human pancreatic duct epithelial cells (HPDEs). By immunostaining, overexpression of PKCα and downregulation of claudin-1 were observed in poorly differentiated human pancreatic cancer tissues and the pancreatic cancer cell line PANC-1. Treatment with the PKCα inhibitor Gö6976 transcriptionally decreased Snail and increased claudin-1 in PANC-1 cells. The PKCα inhibitor prevented upregulation of Snail and downregulation of claudin-1 during EMT induced by transforming growth factor-β1 (TGF-β1) treatment and under hypoxia in PANC-1 cells. The effects of the PKCα inhibitor were in part regulated via an extracellular signal-regulated kinase (ERK) signaling pathway. The PKCα inhibitor also prevented downregulation of the barrier function and fence function during EMT in well-differentiated pancreatic cancer cell line HPAC. In normal HPDEs, the PKCα inhibitor transcriptionally induced not only claudin-1 but also claudin-4, -7 and occludin without a change of Snail. Treatment with the PKCα inhibitor in normal HPDEs prevented downregulation of claudin-1 and occludin by TGF-β1 treatment and enhanced upregulation of claudin-1, -4, -7 and occludin under hypoxia. These findings suggest that PKCα regulates claudin-1 via Snail- and mitogen-activated protein kinase/ERK-dependent pathways during EMT in pancreatic cancer. Thus, PKCα inhibitors may be potential therapeutic agents against the malignancy of human pancreatic cancer cells.

DOI PubMed

Behavior of tricellulin during destruction and formation of tight junctions under various extracellular calcium conditions

Akira Takasawa, Takashi Kojima, Takafumi Ninomiya, Mitsuhiro Tsujiwaki, Masaki Murata, Satoshi Tanaka, Norimasa Sawada

CELL AND TISSUE RESEARCH ( SPRINGER ) 351 ( 1 ) 73 - 84 2013.01 [Refereed]

Tricellulin is an important component of tricellular tight junctions (TJs) and is involved in the formation of tricellular contacts. However, little is known about its regulation during the assembly and disassembly of tricellular TJs. By using the well-differentiated pancreatic cancer cell line HPAC, which highly expresses tricellulin at tricellular contacts, we have investigated changes in the localization, expression and phosphorylation of tricellulin and in its TJ functions as a barrier and fence during the destruction and formation of TJs induced by changes in the extracellular calcium concentration. During both extracellular Ca2+ depletion caused by EGTA treatment and Ca2+ repletion after Ca2+ starvation, the expression of tricellulin increased in whole lysates and in Triton-X-100-insoluble fractions without any change in its mRNA. The increases in immunoreactivity revealed by Western blotting were prevented by alkaline phosphatase treatment. Immunoprecipitation assays showed that tricellulin was phosphorylated on threonine residues when it increased after Ca2+ depletion and repletion. In the early stage after Ca2+ repletion, tricellulin was expressed not only at tricellular contacts but also in the cytoplasm and at bicellular borders. In confocal laser microscopy, tricellulin was observed at the apical-most regions and basolateral membranes of tricellular contacts after Ca2+ repletion. Knockdown of tricellulin delayed the recovery of the barrier and fence functions after Ca2+ repletion. Thus, the dynamic behavior of tricellulin during the destruction and formation of TJs under various extracellular calcium conditions seems to be closely associated with the barrier and fence functions of TJs.

DOI PubMed

Curcumin prevents replication of respiratory syncytial virus and the epithelial responses to it in human nasal epithelial cells.

Kazufumi Obata, Takashi Kojima, Tomoyuki Masaki, Tamaki Okabayashi, Shinichi Yokota, Satoshi Hirakawa, Kazuaki Nomura, Akira Takasawa, Masaki Murata, Satoshi Tanaka, Jun Fuchimoto, Nobuhiro Fujii, Hiroyuki Tsutsumi, Tetsuo Himi, Norimasa Sawada

PloS one 8 ( 9 ) e70225 2013 [International journal]

The human nasal epithelium is the first line of defense during respiratory virus infection. Respiratory syncytial virus (RSV) is the major cause of bronchitis, asthma and severe lower respiratory tract disease in infants and young children. We previously reported in human nasal epithelial cells (HNECs), the replication and budding of RSV and the epithelial responses, including release of proinflammatory cytokines and enhancement of the tight junctions, are in part regulated via an NF-κB pathway. In this study, we investigated the effects of the NF-κB in HNECs infected with RSV. Curcumin prevented the replication and budding of RSV and the epithelial responses to it without cytotoxicity. Furthermore, the upregulation of the epithelial barrier function caused by infection with RSV was enhanced by curcumin. Curcumin also has wide pharmacokinetic effects as an inhibitor of NF-κB, eIF-2α dephosphorylation, proteasome and COX2. RSV-infected HNECs were treated with the eIF-2α dephosphorylation blocker salubrinal and the proteasome inhibitor MG132, and inhibitors of COX1 and COX2. Treatment with salubrinal, MG132 and COX2 inhibitor, like curcumin, prevented the replication of RSV and the epithelial responses, and treatment with salubrinal and MG132 enhanced the upregulation of tight junction molecules induced by infection with RSV. These results suggest that curcumin can prevent the replication of RSV and the epithelial responses to it without cytotoxicity and may act as therapy for severe lower respiratory tract disease in infants and young children caused by RSV infection.

DOI PubMed

Hop water extract inhibits double-stranded RNA-induced thymic stromal lymphopoietin release from human nasal epithelial cells

Jun Fuchimoto, Takashi Kojima, Naoyuki Kobayashi, Tsuyoshi Ohkuni, Noriko Ogasawara, Tomoyuki Masaki, Kazufumi Obata, Kazuaki Nomura, Atsushi Kondoh, Tatsuro Shigyo, Tetsuo Himi, Norimasa Sawada

AMERICAN JOURNAL OF RHINOLOGY & ALLERGY ( OCEAN SIDE PUBLICATIONS INC ) 26 ( 6 ) 433 - 438 2012.11 [Refereed]

Background: Thymic stromal lymphopoietin (TSLP) acts as a master switch for allergic inflammation and plays a key role in allergic diseases, including allergic rhinitis. Double-stranded RNA (dsRNA) recognized by Toll-like receptor 3 (TLR3) strongly activates TSLP release from human nasal epithelial cells (HNECs). Hop (Humulus lupulus L.) extracts have been shown to have potent pharmacologic effects on inflammation. Methods: To investigate whether a hop water extract (HWE) prevents TSLP release from HNECs, human telomerase reverse transcriptase (hTERT)-transfected HNECs, used as a model of normal HNECs, were pretreated with HWE before treatment with the TLR3 ligand Polyinosine-polycytidylic acid (poly[I:C]). Results: In the hTERT-transfected HNECs, treatment with HWE significantly reduced poly(I:C)-induced production and release of TSLP in a dose-dependent manner, as well as dexamethasone. Treatment with the protein kinase C (PKC) inhibitor GF109203X and NF-kappa B inhibitor IMD-0354 also reduced poly(I:C)-induced TSLP release from hTERT-transfected HNECs. Treatment with HWE efficiently prevented up-regulation of PKC activity by 12-O-tetradecanoyl phorbol-13-acetate but not NF-kappa B activity induced by IL-1 beta in hTERT-transfected HNECs. Conclusion: Our results clearly indicated that HWE inhibited dsRNA-induced production and release of TSLP via a PKC signal pathway in HNECs and it may have potent preventive effects against allergic rhinitis.

DOI

Targeting claudin-4 in human pancreatic cancer.

Takashi Kojima, Daisuke Kyuno, Norimasa Sawada

Expert opinion on therapeutic targets 16 ( 9 ) 881 - 7 2012.09 [Refereed] [International journal]

INTRODUCTION: Pancreatic cancer is one of the most malignant human diseases and there is an urgent need to develop novel diagnostic and therapeutic strategies. Claudin-4, overexpressed in pancreatic cancer and its precursor lesions, is a receptor for Clostridium perfringens enterotoxin (CPE). The cytotoxic effects of CPE and monoclonal antibodies against claudin-4 are useful as novel therapeutic tools for pancreatic cancer. AREAS COVERED: This review describes and discusses the studies targeting claudin-4 in normal human pancreatic duct epithelial (HPDE) cells and cancer cells. EXPERT OPINION: Claudin-4 is in part regulated via a PKCα signal transduction pathway in pancreatic cancer cell lines. PKCα inhibitors may represent potential therapeutic agents against human pancreatic cancer cells by the use of CPE cytotoxicity via claudin-4. The COOH-terminal half fragment of CPE (C-CPE) enhances the effectiveness of clinically relevant chemotherapies and can be used as a carrier for drugs and other bacterial toxins to claudin-4-positive cancer cells. hTERT-HPDE cells, in which the human telomerase reverse transcriptase (hTERT) gene is introduced into normal HPDE cells, may be a useful model of normal HPDE cells not only for physiological regulation of claudin-4 expression but also for developing safer and more effective therapeutic methods targeting claudin-4 in pancreatic cancer.

DOI PubMed

【EMTのUP DATE】EMTと膵癌

及能大輔, 小島隆, 山口洋志, 伊東竜哉, 高澤啓, 村田雅樹, 今村将史, 木村康利, 澤田典均, 平田公一

Surgery Frontier ( (株)メディカルレビュー社 ) 19 ( 3 ) 305 - 310 2012.09

膵癌では、原発巣において腺腔構造を維持していた癌細胞が低分化な紡錘形細胞となって移動し、再び高分化な転移巣を形成しているかのような像がしばしば観察される。このような形態変化は、膵癌の浸潤・転移における上皮間葉移行(epithelial-mesenchymal transition;EMT)の特徴像とされている。膵癌のEMT誘導因子には、さまざまなサイトカインや増殖因子、癌周囲微小環境などが挙げられているが、なかでもTGF-βが中心的な役割を担っていると考えられている。EMT誘導因子はSnail、Slug、ZEB1、SIP1、E2A、Twistなどの転写因子を活性化し、あるいはmicroRNAなどを制御した結果、E-cadherinなどの上皮系マーカーを転写レベルで抑制し、N-cadherinなどの間葉系マーカーの発現を転写レベルで促進させ、癌細胞が細胞間接着を喪失し、高い運動性や浸潤能を獲得し、局所浸潤や他臓器転移をもたらすとされている。膵癌のEMTについての研究は、癌の浸潤・転移のメカニズムの理解につながり、膵癌の新規診断・治療法に寄与するものと思われる。(著者抄録)

Epidermal growth factor modulates claudins and tight junctional functions in ovarian cancer cell lines.

Ogawa M, Kojima T, Someya M, Nomura K, Takasawa A, Murata M, Tanaka S, Saito T, Sawada N

Histochemistry and cell biology ( 2 ) 138 323 - 338 2012.08 [Refereed]

DOI PubMed

A novel autocrine-paracrine loop of TSLP in atopic dermatitis is modulated by p63

Kubo Terufumi, Ye Rui, Ara Shihoko, Matsumiya Hiroshi, Kojima Takashi, Kikuchi Tomoki, Sato Noriyuki, Ichimiya Shingo

JOURNAL OF IMMUNOLOGY 188 2012.05 [Refereed]

Claudin-4-targeted therapy using Clostridium perfringens enterotoxin for prostate cancer.

Toshihiro Maeda, Masaki Murata, Hideki Chiba, Akira Takasawa, Satoshi Tanaka, Takashi Kojima, Naoya Masumori, Taiji Tsukamoto, Norimasa Sawada

The Prostate 72 ( 4 ) 351 - 60 2012.03 [International journal]

BACKGROUND: Clostridium perfringens enterotoxin (CPE) triggers lysis of epithelial cells through binding to tight-junction proteins claudin-3 (Cldn3) and Cldn4, which are over-expressed in prostate cancer. We investigated the potential of Cldn-targeted therapy using CPE. METHODS: We investigated the expression levels and subcellular localization of Cldn3 and Cldn4 in primary human prostate cancer tissues, human prostate cancer cell lines (22Rv1, DU145, and PC3) and normal human prostate epithelial cells (PrECs). Cytotoxic effects of CPE on these cells were examined by colorimetric assay. We studied whether knockdown of Cldn3 and/or Cldn4 expression using RNA interference influenced CPE-mediated cytotoxicity. The therapeutic effect of CPE was evaluated in PC3 xenografts in athymic mice. RESULTS: Cldn4 and Cldn3 were expressed in primary human prostate cancer tissues, 22Rv1, DU145, and PC3. Cldn4 protein was expressed in PrEC. Cldn4 was distributed along whole cell membranes of the cancer cell lines, whereas it was localized at tight junctions in PrEC. CPE-mediated cytotoxicity was greatly detected in PC3, but was hardly detectable in PrEC. Reduced expression of Cldn4, but not Cldn3, led to remarkable decreases of cytotoxicity in both PC3 and 22Rv1. The injection of CPE around PC3 xenografts significantly suppressed tumor growth. CONCLUSION: CPE-mediated cytotoxicity was observed in human prostate cancer cell lines, but barely detected in normal human PrECs. The cytotoxic effect depended not only on the expression level of Cldn4 protein but also on its subcellular localization. These results suggest that Cldn4-targeted therapy using CPE may be a new treatment for prostate cancer.

DOI PubMed

The role of IL-33 and its receptor ST2 in human nasal epithelium with allergic rhinitis

R. Kamekura, T. Kojima, K. Takano, M. Go, N. Sawada, T. Himi

CLINICAL AND EXPERIMENTAL ALLERGY ( WILEY ) 42 ( 2 ) 218 - 228 2012.02 [Refereed]

Background Interleukin (IL)-33 is a novel member of the IL-1 cytokine family and a ligand for the orphan IL-1 family receptor ST2. The IL-33 induces T helper 2-type inflammatory responses and is considered to play a crucial rule in allergic inflammations, such as asthma and atopic dermatitis. However, the role of IL-33 and its receptor ST2 in allergic rhinitis remains unknown. Objective We investigated expression of IL-33 and ST2 in the nasal epithelium of patients with allergic rhinitis and the mechanisms of the production of cytokines/chemokines induced by treatment with IL-33 using normal human nasal epithelial cells (HNECs) in vitro. Methods Expression of IL-33 and ST2 in normal and allergic rhinitis nasal mucosa was evaluated by reverse transcription-and real-time polymerase chain reactions and immunohistochemical methods. The IL-33 in serum, and IL-8 and GM-CSF were measured by ELISA. For in vitro experiments, HNECs in primary culture were used. Results The IL-33 levels in the sera of patients with allergic rhinitis were significantly higher than that in normal controls. Expression of IL-33 and ST2 was significantly elevated in the epithelium from patients with allergic rhinitis. The IL-33 mRNA in HNECs in vitro was significantly induced by treatment with IFN-gamma and the toll-like receptor 9 ligand ODN2006. The IL-33-induced production of IL-8 and GM-CSF from HNECs in vitro was significantly suppressed by corticosteroid treatment and distinct signal transduction inhibitors of ERK, p38 MAPK, JNK, NF-kappa B and epidermal growth factor receptor. Conclusions and Clinical Relevance The IL-33 and its receptor ST2 play important roles in allergic rhinitis. The IL-33-mediated inflammatory responses via ST2 are regulated by distinct signalling pathways in HNECs and the IL-33/ST2 pathway may provide new therapeutic targets for allergic rhinitis.

DOI

Regulation of tight junctions in human normal pancreatic duct epithelial cells and cancer cells

Takashi Kojima, Norimasa Sawada

BARRIERS AND CHANNELS FORMED BY TIGHT JUNCTION PROTEINS I ( BLACKWELL SCIENCE PUBL ) 1257 85 - 92 2012 [Refereed]

To investigate the regulation of tight junction molecules in normal human pancreatic duct epithelial (HPDE) cells and pancreatic cancer cells, we introduced the human telomerase reverse transcriptase (hTERT) gene into HPDE cells in primary culture and compared them to pancreatic cancer cell lines. The hTERT-transfected HPDE cells were positive for PDE markers and expressed claudin-1, claudin-4, claudin-7, and claudin-18, occludin, tricellulin, marvelD3, JAM-A, zonula occludens (ZO)-1, and ZO-2. The tight junction molecules, including claudin-4 and claudin-18 of normal HPDE cells, were in part regulated via a protein kinase C signal pathway by transcriptional control. In addition, claudin-18 in normal HPDE cells and pancreatic cancer cells was markedly induced by a PKC activator, and claudin-18 in pancreatic cancer cells was also modified by DNA methylation. In the marvel family of normal HPDE cells and pancreatic cancer cells, tricellulin was upregulated via a c-Jun N-terminal kinase pathway, and marvelD3 was downregulated during Snail-induced epithelial-mesenchymal transition.

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Protein kinase Cα inhibitor enhances the sensitivity of human pancreatic cancer HPAC cells to Clostridium perfringens enterotoxin via claudin-4.

Daisuke Kyuno, Takashi Kojima, Tatsuya Ito, Hiroshi Yamaguchi, Mitsuhiro Tsujiwaki, Akira Takasawa, Masaki Murata, Satoshi Tanaka, Koichi Hirata, Norimasa Sawada

Cell and tissue research 346 ( 3 ) 369 - 81 2011.12 [Refereed] [International journal]

Protein kinase C (PKC) is overexpressed in cancer, including pancreatic cancer, compared with normal tissue. Moreover, PKCα is considered one of the biomarkers for the diagnosis of cancers. In several human cancers, the claudin tight junction molecules are abnormally regulated and are thus promising molecular targets for diagnosis and therapy with Clostridium perfringens enterotoxin (CPE). In order to investigate the changes of tight junction functions of claudins via PKCα activation in pancreatic cancer cells, the well-differentiated human pancreatic cancer cell line HPAC, with its highly expressed tight junction molecules and well-developed barrier function, was treated with the PKC activator 12-O-tetradecanoylphorbol 13-acetate (TPA). Treatment with TPA modified the activity of phosphoPKCα and caused an increase of the Snail family members Snail, Slug and Smad-interacting protein 1 and a decrease of E-cadherin. In HPAC cells treated with TPA, downregulation of claudin-1 and mislocalization of claudin-4 and occludin around the nuclei were observed, together with a decrease in the numbers of tight junction strands and an increase in phosphorylation of claudin-4. The barrier function and the cytotoxicity of CPE were significantly decreased on TPA treatment. All such changes after TPA treatment were prevented by inhibitors of panPKC and PKCα. These findings suggest that, in human pancreatic cancer cells, PKCα activation downregulates tight junction functions as a barrier and as a receptor of CPE via the modification of claudin-1 and -4 during epithelial to mesenchymal transition-like changes. PKCα inhibitors might represent potential therapeutic agents against human pancreatic cancer cells by use of CPE cytotoxicity via claudin-4.

DOI PubMed

Downregulation of tight junction-associated MARVEL protein marvelD3 during epithelial-mesenchymal transition in human pancreatic cancer cells.

Takashi Kojima, Akira Takasawa, Daisuke Kyuno, Tatsuya Ito, Hiroshi Yamaguchi, Koichi Hirata, Mitsuhiro Tsujiwaki, Masaki Murata, Satoshi Tanaka, Norimasa Sawada

Experimental cell research 317 ( 16 ) 2288 - 98 2011.10 [Refereed] [International journal]

The novel tight junction protein marvelD3 contains a conserved MARVEL (MAL and related proteins for vesicle trafficking and membrane link) domain like occludin and tricellulin. However, little is yet known about the detailed role and regulation of marvelD3 in normal epithelial cells and cancer cells, including pancreatic cancer. In the present study, we investigated marvelD3 expression in well and poorly differentiated human pancreatic cancer cell lines and normal pancreatic duct epithelial cells in which the hTERT gene was introduced into human pancreatic duct epithelial cells in primary culture, and the changes of marvelD3 during Snail-induced epithelial-mesenchymal transition (EMT) under hypoxia, TGF-β treatment and knockdown of FOXA2 in well differentiated pancreatic cancer HPAC cells. MarvelD3 was transcriptionally downregulated in poorly differentiated pancreatic cancer cells and during Snail-induced EMT of pancreatic cancer cells in which Snail was highly expressed and the fence function downregulated, whereas it was maintained in well differentiated human pancreatic cancer cells and normal pancreatic duct epithelial cells. Depletion of marvelD3 by siRNAs in HPAC cells resulted in downregulation of barrier functions indicated as a decrease in transepithelial electric resistance and an increase of permeability to fluorescent dextran tracers, whereas it did not affect fence function of tight junctions. In conclusion, marvelD3 is transcriptionally downregulated in Snail-induced EMT during the progression for the pancreatic cancer.

DOI PubMed

Effects of Clostridium perfringens enterotoxin via claudin-4 on normal human pancreatic duct epithelial cells and cancer cells.

Hiroshi Yamaguchi, Takashi Kojima, Tatsuya Ito, Daisuke Kyuno, Yasutoshi Kimura, Masafumi Imamura, Koichi Hirata, Norimasa Sawada

Cellular & molecular biology letters 16 ( 3 ) 385 - 97 2011.09 [Refereed] [International journal]

The tight junction protein claudin-4 is frequently overexpressed in pancreatic cancer, and is also a receptor for Clostridium perfringens enterotoxin (CPE). The cytotoxic effects of CPE are thought to be useful as a novel therapeutic tool for pancreatic cancer. However, the responses to CPE via claudin-4 remain unknown in normal human pancreatic duct epithelial (HPDE) cells. We introduced the human telomerase reverse transcriptase (hTERT) gene into HPDE cells in primary culture as a model of normal HPDE cells in vitro. hTERT-HPDE cells treated with or without 10% FBS and pancreatic cancer cell lines PANC-1, BXPC3, HPAF-II and HPAC were treated with CPE. In Western blotting, the expression of claudin-4 protein in hTERT-HPDE cells treated with 10% FBS was as high as it was in all of the pancreatic cancer cell lines. In hTERT-HPDE cells with or without 10% FBS, cytotoxicity was not observed at any concentration of CPE, whereas in all pancreatic cancer cell lines, CPE had a dose-dependent cytotoxic effect. In hTERT-HPDE cells with 10% FBS, claudin-4 was localized in the apical-most regions, where there are tight junction areas, in which in all pancreatic cancer cell lines claudin-4 was found not only in the apical-most regions but also at basolateral membranes. In hTERT-HPDE cells with 10% FBS after treatment with CPE, downregulation of barrier function and claudin-4 expression at the membranes was observed. In HPAC cells, the sensitivity to CPE was significantly decreased by knockdown of claudin-4 expression using siRNA compared to the control. These findings suggest that, in normal HPDE cells, the lack of toxicity of CPE was probably due to the localization of claudin-4, which is different from that of pancreatic cancer cells. hTERT-HPDE cells in this culture system may be a useful model of normal HPDE cells not only for physiological regulation of claudin-4 expression but also for developing safer and more effective therapeutic methods targeting claudin-4 in pancreatic cancer.

DOI PubMed

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Papers - KOJIMA Takashi