Details of a Researcher - Kanai, Takanori

Papers - Kanai, Takanori

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Human CD14⁺ macrophages in intestinal lamina propria exhibit potent antigen-presenting ability

Kamada Nobuhiko, Hisamatsu Tadakazu, Honda Haruki, Kobayashi Taku, Chinen Hiroshi, Kitazume Mina Tokutake, Takayama Tetsuro, Okamoto Susumu, Koganei Kazutaka, Sugita Akira, Kanai Takanori, Hibi Toshifumi

Journal of Immunology 183 ( 3 ) 1724 - 1731 2009.08

ISSN 0022-1767

Intestinal APCs are considered critical in maintaining the balance between the response against harmful pathogens and the induction of tolerance to commensal bacteria and food Ags. Recently, several studies indicated the presence of gut-specific APC subsets, which possess both macrophage and dendritic cell (DC) markers. These unique APC subsets play important roles in gut immunity, especially for immune regulation against commensal bacteria. Herein, we examined a unique macrophage subset, which coexpressed the macrophage (Mφ) marker CD14 and the DC marker CD209 in human intestinal lamina propria (LP). The LP Mφ subset in both normal control subjects or Crohn's disease (CD) patients induced proliferation of naive CD4⁺ T cells as well as monocyte-derived DCs, and it expressed retinoic acid synthetic enzyme retinaldehyde dehydrogenase 2 and retinol dehydrogenase 10, which induced expression of gut homing receptors on T cells in a retinoic acid-dependent manner. Moreover, the LP Mφ subset strongly evoked differentiation of Th1 cells and slightly induced Th17 cells in both normal control subjects and CD patients; the inducing potential was highest in CD patients. In CD patients, Th17, but not Th1, induction by the LP Mφ subset was enhanced in the presence of commensal bacteria Ags. This enhancement was not observed in normal control subjects. The Th17 induction by the LP Mφ subset was inhibited by neutralization of IL-6 and IL-1β, but it was enhanced by blockade of retinoic acid signaling. These observations highlight a role for LPMφ in the enhanced Th1, and potentially in Th17 differentiation, at the inflammatory site of inflammatory bowel diseases. Copyright © 2009 by The American Association of Immunologists, Inc.

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Homeostatic (IL-7) and effector (IL-17) cytokines as distinct but complementary target for an optimal therapeutic strategy in inflammatory bowel disease

Kanai Takanori, Nemoto Yasuhiro, Kamada Nobuhiko, Totsuka Teruji, Hisamatsu Tadakazu, Watanabe Mamoru, Hibi Toshifumi

Current Opinion in Gastroenterology 25 ( 4 ) 306 - 313 2009.07

ISSN 0267-1379

Lippincott Williams & Wilkins.Purpose of review: This review focuses on CD4⁺ T cells involved in the mediation of inflammatory tissue damage in murine models of inflammatory bowel diseases (IBDs). In particular, we describe the distinct roles of the homeostatic cytokine IL-7, which is essential to the maintenance of colitogenic memory CD4⁺ cells, and the newly discovered effector cytokine IL-17. We also discuss the close correlation between colitogenic Th17-type CD4 ⁺ T cells and inducible CD4⁺CD25⁺Foxp3 ⁺ regulatory T cells. Recent findings: IBDs are characterized by wasting and chronic intestinal inflammation induced by many different cytokine-mediated pathways. It is clearly recognized that medical and surgical interventions do not cure Crohn's disease because relapse is the rule after remission. Until a few years ago, IBD was classified into Th1-dependent, that is, Crohn's disease, and Th2-dependent, that is, ulcerative colitis, phenotypes. However, in recent years, it has been shown that new T-cell subclasses, that is, Th17 and regulatory T cells (T_R), exist independently of Th1 and Th2 and that they play a central role in modulating IBD. Summary: The persistence of IL-7-dependent colitogenic memory CD4⁺ T cells is critical to the maintenance of experimental colitis. On the other hand, though Th1 and Th2 colitogenic memory CD4⁺ cells exist, in recent years the central role of IL-17-producing Th17-type cells in IBD has attracted renewed interest. The development of molecularly targeted therapies aimed at a variety of different Th-dependent pathogenic mechanisms may represent a novel approach to IBD therapy. © 2009 Wolters Kluwer Health

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Rank-rankl signaling pathway is critically involved in the function of CD4⁺CD25⁺ regulatory T cells in chronic colitis

Totsuka Teruji, Kanai Takanori, Nemoto Yasuhiro, Tomita Takayuki, Okamoto Ryuichi, Tsuchiya Kiichiro, Nakamura Tetsuya, Sakamoto Naoya, Akiba Hisaya, Okumura Ko, Yagita Hideo, Watanabe Mamoru

Journal of Immunology 182 ( 10 ) 6079 - 6087 2009.05

ISSN 0022-1767

It is now clear that functional CD4⁺CD25⁺ regulatory T (T_R) cells exist as part of the normal immune population and prevent the development of intestinal inflammation. We have recently shown that CD4⁺CD25⁺ T_R cells reside in the intestine and control intestinal homeostasis in humans and mice. In this study, we demonstrate that the TNF family molecule RANKL and its receptor RANK are critically involved in controlling the function of CD4⁺CD25⁺ T _R cells in the intestine. We first found that RANKL was preferentially expressed on both CD4⁺CD25⁺ T_R cells and colitogenic CD4⁺ T cells, whereas RANK was expressed on dendritic cells. Although neutralizing anti-RANKL mAb did not affect T _R activity of CD4⁺CD25⁺ T_R cells to suppress the proliferation of CD4⁺ responder cells in vitro, in vivo administration of anti-RANKL mAb abrogated CD4⁺CD25⁺ T_R cell-mediated suppression of colitis induced by adoptive transfer of CD4⁺CD45RB^high T cells into SCID mice. Interestingly, an adoptive transfer experiment using Ly5.1⁺CD4 ⁺CD45RB^high cells and Ly5.2⁺CD4 ⁺CD25⁺ T_R cells revealed that the ratio of CD4⁺CD25⁺ T_R cells in total CD4⁺ T cells in inflamed mucosa was significantly decreased by anti-RANKL mAb treatment. Consistent with this, the expression of RANK on lamina propria CD11c⁺ cells from colitic mice was significantly increased as compared with that from normal mice, and in vitro treatment with anti-RANKL mAb suppressed the expansion of CD4⁺Foxp3⁺ T_R cells in culture with colitic lamina propria CD11c⁺ cells. Together, these results suggest that the RANK-RANKL signaling pathway is critically involved in regulating the function of CD4⁺CD25⁺ T_R cells in colitis. Copyright © 2009 by The American Association of Immunologists, Inc.

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Signaling pathway via TNF-α/NF-κB in intestinal epithelial cells may be directly involved in colitis-associated carcinogenesis

Onizawa Michio, Nagaishi Takashi, Kanai Takanori, Nagano Ken Ichi, Oshima Shigeru, Nemoto Yasuhiro, Yoshioka Atsushi, Totsuka Teruji, Okamoto Ryuichi, Nakamura Tetsuya, Sakamoto Naoya, Tsuchiya Kiichiro, Aoki Kazuhiro, Ohya Keiichi, Yagita Hideo, Watanabe Mamoru

American Journal of Physiology - Gastrointestinal and Liver Physiology 296 ( 4 ) 2009.04

ISSN 0193-1857

Treatment with anti-TNF-α MAb has been accepted as a successful maintenance therapy for patients with inflammatory bowel diseases (IBD). Moreover, it has been recently reported that blockade of TNF receptor (TNFR) 1 signaling in infiltrating hematopoietic cells may prevent the development of colitis-associated cancer (CAC). However, it remains unclear whether the TNF-α signaling in epithelial cells is involved in the development of CAC. To investigate this, we studied the effects of anti-TNF-α MAb in an animal model of CAC by administration of azoxymethane (AOM) followed by sequential dextran sodium sulfate (DSS) ingestion. We observed that the NF-κB pathway is activated in colonic epithelia from DSS-administered mice in association with upregulation of TNFR2 rather than TNFR1. Immunoblot analysis also revealed that the TNFR2 upregulation accompanied by the NF-κB activation is further complicated in CAC tissues induced in AOM/DSS-administered mice compared with the nontumor area. Such NF-κB activity in the epithelial cells is significantly suppressed by the treatment of MP6-XT22, an anti-TNF-α MAb. Despite inability to reduce the severity of colitis, sequential administration of MP6-XT22 reduced the numbers and size of tumors in association with the NF-κB inactivation. Taken together, present studies suggest that the TNFR2 signaling in intestinal epithelial cells may be directly involved in the development of CAC with persistent colitis and imply that the maintenance therapy with anti-TNF-α MAb may prevent the development of CAC in patients with long-standing IBD. Copyright © 2009 the American Physiological Society.

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Pathology and pathophysiology of inflammatory bowel diseases. Development of inflammatory bowel disease due to disruption of the intestinal immuno-regulatory mechanism

Kanai Takanori, Hisamatsu Tadanori, Watanabe Mamoru, Hibi Toshifumi

Nihon Naika Gakkai zasshi. The Journal of the Japanese Society of Internal Medicine 98 ( 1 ) 12 - 17 2009.01

ISSN 0021-5384

Requirement of notch activation during regeneration of the intestinal epithelia

Okamoto Ryuichi, Tsuchiya Kiichiro, Nemoto Yasuhiro, Akiyama Junko, Nakamura Tetsuya, Kanai Takanori, Watanabe Mamoru

American Journal of Physiology - Gastrointestinal and Liver Physiology 296 ( 1 ) 2009.01

ISSN 0193-1857

Notch signaling regulates cell differentiation and proliferation, contributing to the maintenance of diverse tissues including the intestinal epithelia. However, its role in tissue regeneration is less understood. Here, we show that Notch signaling is activated in a greater number of intestinal epithelial cells in the inflamed mucosa of colitis. Inhibition of Notch activation in vivo using a γ-secretase inhibitor resulted in a severe exacerbation of the colitis attributable to the loss of the regenerative response within the epithelial layer. Activation of Notch supported epithelial regeneration by suppressing goblet cell differentiation, but it also promoted cell proliferation, as shown in in vivo and in vitro studies. By utilizing tetracycline-dependent gene expression and microarray analysis, we identified a novel group of genes that are regulated downstream of Notch1 within intestinal epithelial cells, including PLA2G2A, an antimicrobial peptide secreted by Paneth cells. Finally, we show that these functions of activated Notch1 are present in the mucosa of ulcerative colitis, mediating cell proliferation, goblet cell depletion, and ectopic expression of PLA2G2A, thereby contributing to the regeneration of the damaged epithelia. This study showed the critical involvement of Notch signaling during intestinal tissue regeneration, regulating differentiation, proliferation, and antimicrobial response of the epithelial cells. Thus Notch signaling is a key intracellular molecular pathway for the proper reconstruction of the intestinal epithelia. Copyright © 2009 the American Physiological Society.

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Musashi-1 suppresses expression of Paneth cell-specific genes in human intestinal epithelial cells

Murayama Minekazu, Okamoto Ryuichi, Tsuchiya Kiichiro, Akiyama Junko, Nakamura Tetsuya, Sakamoto Naoya, Kanai Takanori, Watanabe Mamoru

Journal of Gastroenterology 44 ( 3 ) 173 - 182 2009

ISSN 0944-1174

Background: Musashi-1 (Msi-1) is a RNA-binding protein, known as a putative marker of intestinal stem cells (ISCs). However, little is known about the function of Msi-1 within human intestinal epithelial cells (IECs). Thus, the present study aimed to clarify the role of Msi-1 in differentiation and proliferation of IECs. Methods: A human intestinal epithelial cell line stably expressing Msi-1 was established. Proliferation of the established cell lines was measured by bromodeoxyuridine incorporation, whereas differentiation were assessed by reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of lineage-specific genes. Activities of the Notch and Wnt pathways were examined either by reporter assays or expression of downstream target genes. The distribution of Msi-1 and PLA2G2A expression in vivo was determined by immunohistochemistry. Results: Constitutive expression of Msi-1 in IECs had no significant effect on cell proliferation, but suppressed expression of Paneth cell-specific genes, including PLA2G2A. Msi-1 appeared to suppress expression of the PLA2G2A gene at the mRNA level. Analysis of Notch and Wnt pathway activity, however, revealed no significant change upon Msi-1 expression. The expression of Msi-1 and PLA2G2A in vivo was restricted to IECs residing at the lowest part of the human intestinal crypt, but was clearly separated to within basal columnar cells or mature Paneth cells, respectively. Conclusions: Msi-1 suppresses expression of Panethcell-specific genes in IECs, presumably through a pathway independent from Notch or Wnt. These findings suggest Msi-1 is a negative regulator of Paneth cell differentiation, an may contribute to maintain the undifferentiated phenotype of ISCs. © Springer 2009.

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Two flavonoids extracts from Glycyrrhizae radix inhibit in vitro hepatitis C virus replication

Sekine-Osajima Yuko, Sakamoto Naoya, Nakagawa Mina, Itsui Yasuhiro, Tasaka Megumi, Nishimura-Sakurai Yuki, Chen Cheng Hsin, Suda Goki, Mishima Kako, Onuki Yuko, Yamamoto Machi, Maekawa Shinya, Enomoto Nobuyuki, Kanai Takanori, Tsuchiya Kiichiro, Watanabe Mamoru

Hepatology Research 39 ( 1 ) 60 - 69 2009

ISSN 1386-6346

Aim: Traditional herbal medicines have been used for several thousand years in China and other Asian countries. In this study we screened herbal drugs and their purified compounds, using the Feo replicon system, to determine their effects on in vitro HCV replication. Methods: We screened herbal drugs and their purified extracts for the activities to suppress hepatitis C virus (HCV) replication using an HCV replicon system that expressed chimeric firefly luciferase reporter and neomycin phosphotransferase (Feo) genes. We tested extracts and 13 purified compounds from the following herbs: Glycyrrhizae radix; Rehmanniae radix; Paeoniae radix; Artemisiae capillari spica; and Rhei rhizoma. Results: The HCV replication was significantly and dose-dependently suppressed by two purified compounds, isoliquiritigenin and glycycoumarin, which were from Glycyrrhizae radix. Dose-effect analyses showed that 50% effective concentrations were 6.2 ± 1.0 μg/mL and 15.5 ± 0.8 μg/mL for isoliquiritigenin and glycycoumarin, respectively. The MTS assay did not show any effect on cell growth and viability at these effective concentrations, indicating that the effects of the two compounds were specific to HCV replication. These two compounds did not affect the HCV IRES-dependent translation nor did they show synergistic action with interferon-alpha. Conclusion: Two purified herbal extracts, isoliquiritigenin and glycycoumarin, specifically suppressed in vitro HCV replication. Further elucidation of their mechanisms of action and evaluation of in vivo effects and safety might constitute a new anti-HCV therapeutics. © 2009 The Japan Society of Hepatology.

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Persistent retention of colitogenic CD4⁺ memory T cells causes inflammatory bowel diseases to become intractable

Kanai Takanori, Nemoto Yasuhiro, Tomita Takayuki, Totsuka Teruji, Watanabe Mamoru, Hibi Toshifumi

Inflammatory Bowel Diseases 15 ( 6 ) 926 - 934 2009

ISSN 1078-0998

Despite the advent of an age when "malignant" leukemia is cured by bone marrow transplantation, "benign" inflammatory bowel diseases (IBDs) are still intractable life-long diseases. Why is it that once an IBD develops it lasts a long time? We propose that, the same as in the response to vaccination, immune memory T cells that remember the disease are formed in IBDs and, perceiving them as "benign T-cell leukemia"-like lifelong pathology that hematogenously spreads throughout the body, we here propose that the bone marrow itself, which produces large amounts of the survival factor IL-7, is the reservoir for colitogenic CD4⁺ memory T cells responsible for the intractability of IBDs. Copyright © 2008 Crohn's & Colitis Foundation of America, Inc.

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Single-operator double-balloon endoscopy (DBE) is as effective as dual-operator DBE

Araki Akihiro, Tsuchiya Kiichiro, Okada Eriko, Suzuki Shinji, Oshima Shigeru, Yoshioka Sanae, Yoshioka Atsushi, Kanai Takanori, Watanabe Mamoru

Journal of Gastroenterology and Hepatology (Australia) 24 ( 5 ) 770 - 775 2009

ISSN 0815-9319

Background and Study Aims: Double-balloon endoscopy (DBE) is a new device that allows diagnosis and treatment throughout the entire small intestine. Although the originally described method requires two operators, we have recently developed a method to perform DBE by a single operator. We here assessed the clinical usefulness of this one-person method in comparison to the conventional two-person DBE. Patients and Methods: One hundred sixty-two patients (102 men and 60 women, mean age 59 years) underwent 303 DBE procedures. Total observation time, completion rate of total intestinal and colonic observation, lesion-discovery rate, and complication rate were retrospectively compared between the one-person method and the conventional two-person method of DBE. The one-person method consists of the Grip and Pinch technique and Keep (or Hold) and Slide technique. Results: The total observation times were 95.5 ± 35.1 min and 96.7 ± 47.5 min by one-person and two-person antegrade DBE, respectively, and 103 ± 29.8 min and 111 ± 30.1 min by one-person and two-person retrograde DBE, respectively. The completion rate for examination of the entire small intestine was 74.2% in one-person DBE and 76.5% in two-person DBE, respectively. The lesion-discovery rate was 69.0% in one-person DBE and 65.5% in two-person DBE, respectively. No significant differences between two methods were found in all measures. Also, no difference was observed in complication rate of the two methods. Conclusions: The single-operator method for DBE was as efficient as the dual-operator DBE without any higher risk of complications and, therefore, could replace the conventional dual-operator method in the future. © 2009 Journal of Gastroenterology and Hepatology Foundation and Blackwell Publishing Asia Pty Ltd.

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Single-operator method for double-balloon endoscopy

Araki A., Tsuchiya K., Okada E., Suzuki S., Oshima S., Okamoto R., Kanai T., Watanabe Mamoru

Endoscopy 40 ( 11 ) 936 - 938 2008.11

ISSN 0013-726X

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Flagellin stimulation suppresses IL-7 secretion of intestinal epithelial cells

Yoshioka Atsushi, Okamoto Ryuichi, Oshima Shigeru, Akiyama Junko, Tsuchiya Kiichiro, Nakamura Tetsuya, Kanai Takanori, Watanabe Mamoru

Cytokine 44 ( 1 ) 57 - 64 2008.10

ISSN 1043-4666

IL-7 is a cytokine, which regulates development, maintenance and proliferation of T lymphocytes within the human immune system. Production of IL-7 is observed in a sterile environment such as thymus or bone marrow. However, it is also known that intestinal epithelial cells (IECs) residing in close contact with numerous bacterial stimuli also produce IL-7. Here we show that secretion of IL-7 by IECs is significantly suppressed upon stimulation by various bacterial components, including flagellin. Analysis of the intracellular mechanism by which flagellin regulates IL-7 production revealed that flagellin down-regulates expression of the two major transcripts encoding IL-7. Surprisingly, such function of flagellin was independent from the known transcriptional regulation of the IL-7 gene, as no significant change was observed in the transcriptional activity regulated by the previously identified promoter region. As the stability of IL-7 mRNA also remained unchanged upon flagellin stimulation, results suggested the possible involvement of a yet unknown transcriptional regulation of the IL-7 gene. These results describe a novel regulation of IL-7 production by bacterial stimuli, presumably mediated via Toll-like receptors. The present system might contribute to regulate the local lymphocyte pool, in response to the gut luminal or sub-mucosal bacterial abundance. © 2008 Elsevier Ltd. All rights reserved.

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Inducible expression of microRNA-194 is regulated by HNF-1α during intestinal epithelial cell differentiation

Hino Kimihiro, Tsuchiya Kiichiro, Fukao Taro, Kiga Kotaro, Okamoto Ryuichi, Kanai Takanori, Watanabe Mamoru

RNA 14 ( 7 ) 1433 - 1442 2008.07

ISSN 1355-8382

Maintenance of the intestinal epithelium is based on well-balanced molecular mechanisms that confer the stable and continuous supply of specialized epithelial cell lineages from multipotent progenitors. Lineage commitment decisions in the intestinal epithelium system involve multiple regulatory systems that interplay with each other to establish the cellular identities. Here, we demonstrate that the microRNA system could be involved in intestinal epithelial cell differentiation, and that microRNA-194 (miR-194) is highly induced during this process. To investigate this inducible expression mechanism, we identified the genomic structure of the miR-194-2, -192 gene, one of the inducible class of miR-194 parental genes. Furthermore, we identified its transcriptional regulatory region that contains a consensus-binding motif for hepatocyte nuclear factor-1α (HNF-1α), which is well known as a transcription factor to regulate gene expression in intestinal epithelial cells. By chromatin immunoprecipitation assay and luciferase reporter analysis, we revealed that pri-miR-194-2 expression is controlled by HNF-1α, and its consensus binding region is required for the transcription of pri-miR-194-2 in vivo in an intestinal epithelial cell line, Caco-2. Our observations indicate that microRNA genes could be targets of lineage-specific transcription factors and that microRNAs are regulated by a tissue-specific manner in the intestinal epithelium. Therefore, our work suggests that induced expression of these microRNAs have important roles in intestinal epithelium maturation. Published by Cold Spring Harbor Laboratory Press. Copyright © 2008 RNA Society.

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Continuous generation of colitogenic CD4⁺ T cells in persistent colitis

Tomita Takayuki, Kanai Takanori, Fujii Toshimitsu, Nemoto Yasuhiro, Okamoto Ryuichi, Tsuchiya Kiichiro, Totsuka Teruji, Sakamoto Naoya, Watanabe Mamoru

European Journal of Immunology 38 ( 5 ) 1264 - 1274 2008.05

ISSN 0014-2980

Inflammatory bowel diseases take chronic courses due to the expansion of colitogenic CD4⁺ cells. However, it is unclear whether the persistent disease is driven by continuous reactivation of colitogenic memory CD4⁺ cells to generate effector CD4⁺ cells or by continuous generation of effector CD4⁺ cells from naïve cells. To clarify this issue, we performed a series of sequential adoptive transfers of Ly5.2⁺ and Ly5.1⁺ CD4⁺CD45RB^high cells into RAG-2^-/- mice at different time points. We show here that the secondarily transferred CD4⁺CD45RB^high cells can be converted to CD4⁺CD44^highCD62L^-IL-7Rα ^high effector-memory T cells even in the presence of pre-existing effector-memory CD4⁺ cells. Although the total cell numbers of CD4⁺ cells in established colitic mice were consistently equivalent irrespective of the number of primarily transferred cells, the ratio of primarily and secondarily transferred cells was dependent on the ratio of the transferred cell numbers, but not on the order of the transfer. Of note, we found that primarily transferred CD4⁺ cells produced significantly lower amounts of IFN-γ and IL-17 than CD4⁺ cells arising from secondary transfer. In conclusion, the continuous generation of colitogenic CD4⁺ cells that compensate for exhausted CD4⁺ cells may be one of the mechanisms involved in the persistence of colitis. © 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

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Immunosenescent colitogenic CD4+ T cells convert to regulatory cells and suppress colitis

Totsuka Teruji, Kanai Takanori, Nemoto Yasuhiro, Tomita Takayuki, Tsuchiya Kiichiro, Sakamoto Naoya, Okamoto Ryuichi, Watanabe Mamoru

European Journal of Immunology 38 ( 5 ) 1275 - 1286 2008.05

ISSN 0014-2980

Inflammatory bowel diseases progress steadily by the expansion of colitogenic CD4⁺ cells. However, it remains unknown whether colitogenic CD4⁺ cells are long-living like memory cells or exhausted like effector cells. To assess the longevity of colitogenic lamina propria (LP) CD4⁺ cells, we performed sequential transfers of LP CD4⁺ cells from colitic CD4⁺CD45RB^high cell-transferred SCID mice into new SCID mice. Although SCID mice transferred with colitic LP CD4⁺ cells stably developed colitis until at least the sixth transfer, the interval to the development of colitis gradually lengthened as the number of transfers increased. The incidence of colitis gradually decreased after the seventh transfer. Furthermore, non-colitic LP CD4⁺ cells from mice transferred over seven times expressed significantly higher levels of PD-1 and produced significantly lower amounts of IFN-γ, TNF-α, and IL-17 than colitic LP CD4⁺ cells recovered after the first transfer. Most notably, we found that re-transfer of non-colitic LP CD4⁺ cells recovered after multiple transfers prevented the development of colitis in SCID mice co-transferred with CD4⁺CD45RB^high cells. Thus, colitogenic LP CD4⁺ cells may be exhausted over time, become non-functional, convert to regulatory cells, and finally suppress colitis in the process of immunosenescence. © 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

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Proteasomal degradation of Atoh1 by aberrant Wnt signaling maintains the undifferentiated state of colon cancer

Aragaki Mikayo, Tsuchiya Kiichiro, Okamoto Ryuichi, Yoshioka Sanae, Nakamura Tetsuya, Sakamoto Naoya, Kanai Takanori, Watanabe Mamoru

Biochemical and Biophysical Research Communications 368 ( 4 ) 923 - 929 2008.04

ISSN 0006-291X

Atoh1 plays a crucial role in intestinal cell differentiation. We have demonstrated that its human homolog Hath1 protein is targeted by the Wnt-GSK3 axis, resulting in the proteasomal degradation in human colon cancer. However, the contribution of Hath1 degradation to the undifferentiated state of colon cancer remains unknown. In this study, we demonstrated that both constitutive expression of mutant Hath1 and stabilization of Hath1 protein by a GSK3 inhibitor in colon cancer cells increased the expression of MUC2 known as a representative function of differentiated goblet cells. This means that Hath1 protein degradation may be required for maintaining the undifferentiated state of colon cancers, and that GSK3 inhibitors have potential for use in cancer therapy. © 2008 Elsevier Inc. All rights reserved.

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MyD88-dependent pathway in T cells directly modulates the expansion of colitogenic CD4⁺ T cells in chronic colitis

Tomita Takayuki, Kanai Takanori, Fujii Toshimitsu, Nemoto Yasuhiro, Okamoto Ryuichi, Tsuchiya Kiichiro, Totsuka Teruji, Sakamoto Naoya, Akira Shizuo, Watanabe Mamoru

Journal of Immunology 180 ( 8 ) 5291 - 5299 2008.04

ISSN 0022-1767

TLRs that mediate the recognition of pathogen-associated molecular patterns are widely expressed on/in cells of the innate immune system. However, recent findings demonstrate that certain TLRs are also expressed in conventional TCRαβ⁺ T cells that are critically involved in the acquired immune system, suggesting that TLR ligands can directly modulate T cell function in addition to various innate immune cells. In this study, we report that in a murine model of chronic colitis induced in RAG-2^-/- mice by adoptive transfer of CD4⁺CD45RB^high T cells, both CD4⁺CD45RB^high donor cells and the expanding colitogenic lamina propria CD4⁺CD44^hlgh memory cells expresses a wide variety of TLRs along with MyD88, a key adaptor molecule required for signal transduction through TLRs. Although RAG-2^-/- mice transferred with MyD88^-/-CD4⁺CD45RB^high cells developed colitis, the severity was reduced with the delayed kinetics of clinical course, and the expansion of colitogenic CD4⁺ T cells was significantly impaired as compared with control mice transferred with MyD88^+/+CD4 ⁺CD45RB^high cells. When RAG-2^-/- mice were transferred with the same number of MyD88^+/+ (Ly5.1⁺) and MyDSS^-/- (Ly5.2⁺) CD4⁺CD45RB^high cells, MyD88^-/-CD4 ⁺ T cells showed significantly lower proliferative responses assessed by in vivo CFSE division assay, and also lower expression of antiapoptotic Bcl-2/Bcl-x_L molecules and less production of IFN-γ and IL-17, compared with the paired MyD88 ^+/+CD4⁺ T cells. Collectively, the MyD88-dependent pathway that controls TLR signaling in T cells may directly promote the proliferation and survival of colitogenic CD4⁺ T cells to sustain chronic colitis. The Journal of Immunology, 2008, 180: Copyright © 2008 by The American Association of Immunologists, Inc.

Development of plaque assays for hepatitis C virus-JFH1 strain and isolation of mutants with enhanced cytopathogenicity and replication capacity

Sekine-Osajima Yuko, Sakamoto Naoya, Mishima Kako, Nakagawa Mina, Itsui Yasuhiro, Tasaka Megumi, Nishimura-Sakurai Yuki, Chen Cheng Hsin, Kanai Takanori, Tsuchiya Kiichiro, Wakita Takaji, Enomoto Nobuyuki, Watanabe Mamoru

Virology 371 ( 1 ) 71 - 85 2008.02

ISSN 0042-6822

HCV culture in vitro results in massive cell death, which suggests the presence of HCV-induced cytopathic effects. Therefore, we investigated its mechanisms and viral nucleotide sequences involved in this effect using HCV-JFH1 cell culture and a newly developed HCV plaque assay technique. The plaque assay developed cytopathic plaques, depending on the titer of the inoculum. In the virus-infected cells, the ER stress markers, GRP78 and phosphorylated eIF2-alpha, were overexpressed. Cells in the plaques were strongly positive for an apoptosis marker, annexin V. Isolated virus subclones from individual plaque showed greater replication efficiency and cytopathogenicity than the parental virus. The plaque-purified virus had 9 amino acid substitutions, of which 5 were clustered in the C terminal of the NS5B region. Taken together, the cytopathic effect of HCV infection involves ER-stress-induced apoptotic cell death. Certain HCV genomic structures may determine the viral replication capacity and cytopathogenicity. © 2007 Elsevier Inc. All rights reserved.

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Systemic, but not intestinal, IL-7 is essential for the persistence of chronic colitis

Tomita Takayuki, Kanai Takanori, Nemoto Yasuhiro, Totsuka Teruji, Okamoto Ryuichi, Tsuchiya Kiichiro, Sakamoto Naoya, Watanabe Mamoru

Journal of Immunology 180 ( 1 ) 383 - 390 2008.01

ISSN 0022-1767

We previously demonstrated that IL-7 is produced by intestinal goblet cells and is essential for the persistence of colitis. It is well known, however, that goblet cells are decreased or depleted in the chronically inflamed mucosa of animal colitis models or human inflammatory bowel diseases. Thus, in this study, we assess whether intestinal IL-7 is surely required for the persistence of colitis using a RAG-1/2^-/- colitis model induced by the adoptive transfer of CD4⁺CD45RB^high T cells in combination with parabiosis system. Surprisingly, both IL-7^-/- x RAG-1^-/- and IL-7^+/+ x RAG-1^-/- host mice developed colitis 4 wk after parabiosis to a similar extent of colitic IL-7^+/+ x RAG-1 ^-/- donor mice that were previously transferred with CD4 ⁺CD45RB^high T cells. Of note, although the number of CD4⁺ T cells recovered from the spleen or the bone marrow of IL-7^-/- x RAG-1^-/- host mice was significantly decreased compared with that of IL-7^+/+ x RAG-1^-/- host mice, an equivalent number of CD4⁺ T cells was recovered from the lamina propria of both mice, indicating that the expansion of CD4⁺ T cells in the spleen or in the bone marrow is dependent on IL-7, but not in the lamina propria. Development of colitis was never observed in parabionts between IL-7^+/+ x RAG-1^-/- host and noncolitic IL-7^-/- x RAG-1^-/- donor mice that were transferred with CD4 ⁺CD45RB^high T cells. Collectively, systemic, but not intestinal, IL-7 is essential for the persistence of colitis, suggesting that therapeutic approaches targeting the systemic IL-7/IL-7R signaling pathway may be feasible in the treatment of inflammatory bowel diseases. Copyright © 2007 by The American Association of Immunologists, Inc.

Current pharmacologic therapies and emerging alternatives in the treatment of ulcerative colitis

Nakamura Tetsuya, Nagahori Masakazu, Kanai Takanori, Watanabe Mamoru

Digestion 77 ( SUPPL. 1 ) 36 - 41 2008.01

ISSN 0012-2823

Recent progress in both basic and clinical research has led us to develop sophisticated and effective medical therapy of ulcerative colitis (UC). Although classical agents such as aminosalicylates, corticosteroids and immunomodulatory drugs have remained as the gold standard for decades, their novel formulations and/or dosage regimens have changed their placements in the medical management of UC. In addition, studies have shown that a number of novel therapeutic agents, designed to target specific mechanisms involved in the inflammatory cascade, have efficacy for the treatment of UC and they will have significant clinical impacts in the near future. A clear understanding of the proven and potential benefits of both the standard and emerging therapies will be required for the optimum individual care of patients with varied clinical presentations. Copyright © 2008 S. Karger AG.

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