Details of a Researcher - KOJIMA Takashi

Papers - KOJIMA Takashi

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A novel monoclonal antibody against the second extracellular loop of occludin disrupts epithelial cell polarity.

Tokunaga Y, Kojima T, Osanai M, Murata M, Chiba H, Tobioka H, Sawada N

The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society ( 7 ) 55 735 - 744 2007.07 [Refereed]

DOI PubMed

Connexins induce and maintain tight junctions in epithelial cells.

Kojima T, Murata M, Go M, Spray DC, Sawada N

The Journal of membrane biology ( 1-3 ) 217 13 - 19 2007.06 [Refereed]

DOI PubMed

Oncostatin M induces upregulation of claudin-2 in rodent hepatocytes coinciding with changes in morphology and function of tight junctions.

Imamura M, Kojima T, Lan M, Son S, Murata M, Osanai M, Chiba H, Hirata K, Sawada N

Experimental cell research ( 9 ) 313 1951 - 1962 2007.05 [Refereed]

DOI PubMed

The nuclear receptor hepatocyte nuclear factor 4 alpha acts as a morphogen to induce the formation of microvilli

Hideki Chiba, Naoyuki Sakai, Masaki Murata, Makoto Osanai, Takafumi Ninomiya, Takashi Kojima, Norimasa Sawada

JOURNAL OF CELL BIOLOGY ( ROCKEFELLER UNIV PRESS ) 175 ( 6 ) 971 - 980 2006.12 [Refereed]

Microvilli are actin-based organelles found on apical plasma membranes that are involved in nutrient uptake and signal transduction. Numerous components, including ezrin/radixin/moesin (ERM) proteins, have been identified that link filamentous actins to transmembrane proteins, but the signals driving microvillus biogenesis are not known. In this study, we show that the conditional and/or ectopic expression of a nuclear receptor, hepatocyte nuclear factor 4 alpha (HNF4 alpha), triggers microvillus morphogenesis. We also demonstrate that HNF4 alpha expression induces ERM-binding phosphoprotein 50 (EBP50) expression and that attenuation of EBP50 using RNA interference inhibits microvillus development. We conclude that HNF4 alpha acts as a morphogen to trigger microvillus formation.

DOI PubMed

Connexin 26 expression prevents down-regulation of barrier and fence functions of tight junctions by Na+/K+-ATPase inhibitor ouabain in human airway epithelial cell line Calu-3.

Go M, Kojima T, Takano K, Murata M, Koizumi J, Kurose M, Kamekura R, Osanai M, Chiba H, Spray DC, Himi T, Sawada N

Experimental cell research ( 19 ) 312 3847 - 3856 2006.11 [Refereed]

DOI PubMed

Phosphorylation of ezrin enhances microvillus length via a p38 MAP-kinase pathway in an immortalized mouse hepatic cell line.

Lan M, Kojima T, Murata M, Osanai M, Takano K, Chiba H, Sawada N

Experimental cell research ( 2 ) 312 111 - 120 2006.01 [Refereed]

DOI PubMed

p53関連分子による胸腺ストローマの細胞間結合制御

小柴茂, 一宮慎吾, 小島隆, 外岡暁子, 村田雅樹, 木村幸子, 横山繁昭, 氷見徹夫, 佐藤昇志

日本免疫学会総会・学術集会記録 ( (NPO)日本免疫学会 ) 35 65 - 65 2005.11

Behavior of tight-junction, adherens-junction and cell polarity proteins during HNF-4 alpha-induced epithelial polarization

S Satohisa, H Chiba, M Osanai, S Ohno, T Kojima, T Saito, N Sawada

EXPERIMENTAL CELL RESEARCH ( ELSEVIER INC ) 310 ( 1 ) 66 - 78 2005.10 [Refereed]

We previously reported that expression of tight-junction molecules occludin, claudin-6 and claudin-7, as well as establishment of epithelial polarity, was triggered in mouse F9 cells expressing hepatocyte nuclear factor (HNF)-4 alpha [H. Chiba, T. Gotoh, T. Kojima, S. Satohisa, K. Kikuchi, M. Osanai, N. Sawada. Hepatocyte nuclear factor (HNF)-4a triggers formation of functional tight junctions and establishment of polarized epithelial morphology in F9 embryonal carcinoma cells, Exp. Cell Res. 286 (2003) 288-297]. Using these cells, we examined in the present study behavior of tight-junction, adherens-junction and cell polarity proteins and elucidated the molecular mechanism behind HNF-4 alpha-initiated junction formation and epithelial polarization. We herein show that not only ZO-1 and ZO-2, but also ZO-3, junctional adhesion molecule (JAM)-B, JAM-C and cell polarity proteins PAR-3, PAR-6 and atypical protein kinase C (aPKC) accumulate at primordial adherens junctions in undifferentiated F9 cells. In contrast, CRB3, Pals1 and PATJ appeared to exhibit distinct subcellular localization in immature cells, Induced expression of HNF-4 alpha led to translocation of these tight-junction and cell polarity proteins to beltlike tight junctions, where occludin, claudin-6 and claudin-7 were assembled, in differentiated cells. Interestingly, PAR-6, aPKC, CRB3 and Pals1, but not PAR-3 or PATJ, were also concentrated on the apical membranes in differentiated cells. These findings indicate that HNF-4 alpha provokes not only expression of tight-junction adhesion molecules, but also modulation of subcellular distribution of junction and cell polarity proteins, resulting in junction formation and epithelial polarization. (c) 2005 Elsevier Inc. All rights reserved.

DOI

Down-regulation of survival signaling through MAPK and Akt in occludin-deficient mouse hepatocytes in vitro.

Murata M, Kojima T, Yamamoto T, Go M, Takano K, Osanai M, Chiba H, Sawada N

Experimental cell research ( 1 ) 310 140 - 151 2005.10 [Refereed]

DOI PubMed

胸腺ストローマの細胞間結合とその制御

小柴茂, 一宮慎吾, 小島隆, 村田雅樹, 外岡暁子, 木村幸子, 横山繁昭, 氷見徹夫, 佐藤昇志

日本病理学会会誌 ( (一社)日本病理学会 ) 94 ( 1 ) 367 - 367 2005.03

胸腺スカフォールドの組織構築とその制御

小柴茂, 一宮慎吾, 小島隆, 近藤伸彦, 外岡暁子, 村田雅樹, 高桑麗子, 横山繁昭, 氷見徹夫, 佐藤昇志

日本免疫学会総会・学術集会記録 ( (NPO)日本免疫学会 ) 34 72 - 72 2004.11

Role of the p38 MAP-kinase signaling pathway for Cx32 and claudin-1 in the rat liver

Takashi Kojima, Toshinobu Yamamoto, Masaki Murata, Mengdong Lan, Ken-Ichi Takano, Mitsuru Go, Shingo Ichimiya, Hideki Chiba, Norimasa Sawada

Cell Communication and Adhesion 10 ( 4-6 ) 437 - 443 2003.07 [Refereed]

Liver regeneration and cholestasis are associated with adaptive changes in expression of gap and tight junctions through signal transduction. The roles of stress responsitive MAP-kinase, p38 MAP-kinase, in the signaling pathway for gap junction protein, Cx32, and tight junction protein, claudin-1, were examined in rat liver in vivo and in vitro, including regeneration following partial hepatectomy and cholestasis after common bile duct ligation. Changes in the expression and function of Cx32 and claudin-1 in hepatocytes in vivo were studied using the p38 MAP-kinase inhibitor SB203580. Following partial hepatectomy and common bile duct ligation, down-regulation of Cx32 protein was inhibited by SB203580 treatment. Up-regulation of claudin-1 protein was enhanced by SB203580 treatment after partial hepatectomy but not common bile duct ligation. However, no change of the Ki-67 labeling index (which is a marker for cell proliferation) in the livers treated with SB203580, was observed compared to that without SB203580 treatment. In primary cultures of rat hepatocytes, however, treatment with a p38 MAP-kinase activator, anisomycin, decreased Cx32 and claudin-1 protein levels. p38 MAP-kinase may be an important signaling pathway for regulation of gap and tight junctions in hepatocytes. Changes of gap and tight junctions during liver regeneration and cholestasis are shown to be in part controlled via the p38 MAP-kinase signaling pathway and are independent of cell growth.

PubMed

Expression of p21(waf-1/cip-1) is significantly induced in the livers of LEC rats with chronic liver injury

N Sawada, T Kojima, H Obata, H Isomura, S Atsumi, M Sawaki, N Tsuzuki, H Tobioka, Y Kokai, M Satoh, M Mori

JAPANESE JOURNAL OF CANCER RESEARCH ( JAPANESE CANCER ASSOCIATION ) 87 ( 11 ) 1102 - 1105 1996.11 [Refereed]

It is reported that hepatocytes isolated from LEC rats with chronic liver injury show reduced growth activity in primary culture. To elucidate the molecular basis of this phenomenon, we examined expression of p21(waf-1/cip-1) and p27, cyclin-dependent kinase inhibitors, by northern blot analysis. The expression of p21(waf-1/cip-1) in the LEC rat liver was 3-fold higher than that of age-matched SD rat liver, while there was no significant difference in p27 expression level. Western blot analysis also revealed a significant increase in p21(waf-1/cip-1) in the nuclear matrix fraction of the LEC rat liver. Immunohistochemically, p21(waf-1/cip-1) was detected in the nuclei of normal LEC rat hepatocytes, but not in those of hepatocellular carcinoma cells, suggesting selective growth of neoplastic hepatocytes.

p21(waf-1/cip-1/sdi-1) is expressed at G1 phase in primary culture of hepatocytes from old rats, presumably preventing the cells from entering the S phase of the cell cycle

N Sawada, T Kojima, H Obata, M Saitoh, H Isomura, Y Kokai, M Satoh, M Mori

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ( ACADEMIC PRESS INC JNL-COMP SUBSCRIPTIONS ) 228 ( 3 ) 819 - 824 1996.11 [Refereed]

To elucidate whether p21(waf-1/cip-1/sdi-1) expression is associated with loss of growth potential of hepatocytes of old rats, we determined p21(waf-1/cip-1/sdi-1) expression of hepatocytes from old (30 months) rats during the cell cycle in primary culture. A high level of expression oi p21(waf-1/cip-1/sdi-1) was detected at the G1 phase in old-rat hepatocytes, but after the S phase in young-rat hepatocytes. Consistently, the incidence of the cells positive for p21(waf-1/cip-1/sdi-1) in nuclei before entering the S phase was significantly higher in old-rat hepatocytes than in young-rat hepatocytes. These results account for the loss of growth potential of old-rat hepatocytes in vitro and the marked retardation of regeneration of liver in old rats in vivo. (C) 1996 Academic Press, Inc.

Changes in cellular distribution of connexins 32 and 26 during formation of gap junctions in primary cultures of rat hepatocytes

T Kojima, M Yamamoto, H Tobioka, T Mizuguchi, T Mitaka, Y Mochizuki

EXPERIMENTAL CELL RESEARCH ( ACADEMIC PRESS INC JNL-COMP SUBSCRIPTIONS ) 223 ( 2 ) 314 - 326 1996.03 [Refereed]

In the adult rat hepatocyte, gap junction proteins consist of connexin 32 (Cx32) and connexin 26 (Cx26), Previously, we reported that both Cx32 and Cx26 were markedly induced and maintained in primary cultures of adult rat hepatocytes, The reappearing gap junctions were accompanied by increases in both the proteins and the mRNAs, and they were well maintained together with extensive gap junctional intercellular communication (GJIC) for more than 4 weeks. In the present study, we examined the cellular location of the gap junction proteins and the structures in the hepatocytes cultured in our system, using confocal laser microscopy and immunoelectron microscopy of cells processed for Cx32 and Cx26 immunocytochemistry and freeze-fracture analysis, In immunoelectron microscopy, the size of Cx32-immunoreactive gap junction structures on the plasma membrane increased with time of culture, and some of them were larger than those in liver sections in vivo. Freeze-fracture analysis also showed that the size of gap junction plaques increased and that the larger gap junction plaques were composed of densely packed particles. These results suggest that in this culture system, not only the synthesis of Ox proteins but also the size of the gap junction plaques was increased markedly. In the adluminal lateral membrane of the cells, Cx32-immunoreactive lines were observed and many small gap junction plaques were closely associated with a more developed tight junction network. In the basal region of the cells, small Cx32- and Cx26-immunoreactive dots were observed in the cytoplasm and several annular structures labeled with the antibody to Cx32 were observed in the cytoplasm, These results indicated the formation and degradation of gap junctions in the cultured hepatocytes. (C) 1996 Academic Press, Inc.

Effects of oxygen radical scavengers on connexins 32 and 26 expression in primary cultures of adult rat hepatocytes

T Kojima, T Mitaka, T Mizuguchi, Y Mochizuki

CARCINOGENESIS ( OXFORD UNIV PRESS UNITED KINGDOM ) 17 ( 3 ) 537 - 544 1996.03 [Refereed]

Although we recently reported our success in inducing and maintaining the gap junction proteins connexin 26 (Cx26) and connexin 32 (Cx32) in adult rat hepatocytes cultured in serum-free L-15 medium supplemented with epidermal growth factor, dimethylsulfoxide (DMSO) and glucagon, the mechanisms by which DMSO induces gap junctions are still not clear, It is known that DMSO is not only a differentiation reagent for various cells but also a powerful scavenger of oxygen radicals, In the present study, by using this culture system and the measurement of oxidative stress by the nitro blue tetrazolium (NBT) formazan assay, we have examined the effect of oxygen radical scavengers such as DMSO, dimethylthiourea (DMTU) and alpha-tocopherol on the expression of both Cxs and on gap junctional intercellular communication (GJIC), as compared to another differentiation reagent, hexamethylene-bis-acetamide (HMBA), DMSO and DMTU clearly inhibited the oxidative stress of the cultured hepatocytes, while alpha-tocopherol and HMBA did not, The expression of Cx26 and Cx32 in the cultured hepatocytes was markedly induced by DMSO and DMTU, Furthermore, extensive GJIC was also observed with DMSO and DMTU, These results suggest that the expression of gap junctions in the hepatocytes may be closely related to oxidative stress and that oxygen radical scavengers may be important substances in inducing this expression.

INDUCTION AND REGULATION OF CONNEXIN26 BY GLUCAGON IN PRIMARY CULTURES OF ADULT-RAT HEPATOCYTES

T KOJIMA, T MITAKA, Y SHIBATA, Y MOCHIZUKI

JOURNAL OF CELL SCIENCE ( COMPANY OF BIOLOGISTS LTD ) 108 2771 - 2780 1995.08 [Refereed]

In the adult rat hepatocyte, the gap junction proteins consist of a major component, connexin32 (Cx32) and a minor component, connexin26 (Cx26). Although we recently reported our success in inducing and maintaining Cx32 in adult rat hepatocytes cultured in serum-free L-15 medium supplemented with epidermal growth factor and 2% dimethyl sulfoxide, it was very difficult to induce Cx26 in the primary hepatocytes. In the present study, we found that the addition of 10(-7) M glucagon into the culture medium could dramatically induce Cx26 mRNA and protein. Although the expression of Cx32 mRNA was also influenced by glucagon, the increase of the expression was small. Immunocytochemically, Cx26-positive spots were observed between most adjacent cells and were co-localized with the Cx32-positive spots. We also examined whether 0.5 mM dibutyl cyclic AMP could induce expression of Cx26 in the cells. The effect of dexamethasone on the expression of Cx26 mRNA compared to that of Cx32 mRNA was examined. For the induction and maintenance of Cx26 mRNA, more than 10(-7) M dexamethasone was necessary in this culture. These results suggest that expression of Cx26 in hepatocytes may be regulated by the concentrations of glucagon and glucocorticoid hormones.

REAPPEARANCE AND LONG-TERM MAINTENANCE OF CONNEXIN32 IN PROLIFERATED ADULT-RAT HEPATOCYTES - USE OF SERUM-FREE L-15 MEDIUM SUPPLEMENTED WITH EGF AND DMSO

T KOJIMA, T MITAKA, DL PAUL, M MORI, Y MOCHIZUKI

JOURNAL OF CELL SCIENCE ( COMPANY OF BIOLOGISTS LTD ) 108 1347 - 1357 1995.04 [Refereed]

Intercellular communication, especially gap junctional communication, is thought to be one of the highly differentiated functions of hepatocytes. In primary cultures of rat hepatocytes, it has been considered that the maintenance and the reinduction of differentiated functions is very difficult. In the present study, we succeeded in inducing the gap junctional protein connexin32 (Cx32) in adult rat hepatocytes cultured in serum-free L-15 medium supplemented with epidermal growth factor (EGF) and dimethylsulfoxide (DMSO), When the hepatocytes were cultured in L-15 medium supplemented with 20 mM NaHCO3 and 10 ng/ml EGF in a 5% CO2:95% air incubator, the cells proliferated, Fluorescence immunocytochemistry showed spots immunoreactive to Cx32 on the cell membranes between adjacent cells until day 3, but only a few Cx32-positive spots were found after day 4. Western and northern blot analyses also showed that the amounts of both the protein and mRNA of Cx32 in the cells decreased with time in culture, However, when the cells were treated with 2% DMSO from day 4, the immunoreactive spots reappeared on the cell membranes from day 6 and both their number and intensity gradually increased, The reappearance of Cx32 was accompanied by increases in both the protein and mRNA of Cx32, Futhermore, the expression of Cx32 was well maintained, together with extensive gap junctional intercellular communication, for more than 4 weeks, In addition, ultrastructually, many gap junctional structures were observed between the hepatocytes, and the antibodies to Cx32 were shown to bind to those structures, This culture system may be useful for studies of the reconstruction of the gap junctional structure, the intracellular pathways of the proteins, and the regulation of synthesis and processing in differentiated hepatocytes.

RAPID APPEARANCE OF CONNEXIN 26-POSITIVE GAP-JUNCTIONS IN CENTRILOBULAR HEPATOCYTES WITHOUT INDUCTION OF MESSENGER-RNA AND PROTEIN-SYNTHESIS IN ISOLATED-PERFUSED LIVER OF FEMALE RAT

T KOJIMA, N SAWADA, M OYAMADA, H CHIBA, H ISOMURA, M MORI

JOURNAL OF CELL SCIENCE ( COMPANY OF BIOLOGISTS LTD ) 107 3579 - 3590 1994.12

In the adult rat liver, the gap junction protein connexin 32 (Cx32) is evenly distributed in hepatocytes within the liver lobules, while connexin 26 (Cx26) is: preferentially localized in hepatocytes in periportal zones. We report here that Cx26-positive gap junctions rapidly appear in the centrilobular hepatocytes of adult female rat livers during a 30 minute perfusion of the liver through the hepatic portal vein with a 1:1 mixture of Dulbecco's modified Eagle's medium (DMEM) and oxygen transport FC-43 fluid at a physiological flow rate without any changes in the distribution of Cx32. The change in the localization of Cx26 was closely related to that of E-cadherin, and there was no significant increase in the amounts of Cx26 protein and mRNA. The appearance of Cx26 in the centrilobular hepatocytes was inhibited by treatment with cytoskeleton disrupters such as colchicine and cytochalasin B, and intracytoplasmic transport inhibitors such as brefeldin A. The liver perfusion induced the appearance of Cx26 in the centrilobular hepatocytes only in female rats. Estrogen treatment of ovariectomized rats caused the appearance of both Cx26 and E-cadherin in centrilobular hepatocytes not only in the perfused liver but also in the non-perfused liver. Our results indicate that in the rat liver: (a) the localization of Cx26 can be modulated by a post-translational mechanism; (b) E-cadherin may play an important role in the formation of gap junctions composed of Cx26; and (c) the formation of gap junctions is regulated by female steroid hormones.

PubMed

SEQUENTIAL-CHANGES IN INTERCELLULAR-JUNCTIONS BETWEEN HEPATOCYTES DURING THE COURSE OF ACUTE LIVER-INJURY AND RESTORATION AFTER THIOACETAMIDE TREATMENT

T KOJIMA, N SAWADA, Y ZHONG, M OYAMADA, M MORI

VIRCHOWS ARCHIV-AN INTERNATIONAL JOURNAL OF PATHOLOGY ( SPRINGER VERLAG ) 425 ( 4 ) 407 - 412 1994.11

Sequential changes of gap junctions (GJs), tight junctions (TJs) and desmosomes (DSs) between hepatocytes during restorative proliferation were studied in rats after a single intraperitoneal administration of 200 mg/kg thioacetamide (TAA). Antibody against connexin 32 was used to demonstrate GJs; simultaneously the changes in TJs and DSs were studied using antibodies against 7H6 protein and desmoplakins. Propidium iodide and bromodeoxyuridine were used to recognize necrotic and proliferative cells. GJs were evenly distributed in early necrotic hepatocytes at 16 h after TAA treatment, then disappeared from necrotic and surrounding cells at 24 h. At 48 h, GJs had disappeared completely from hepatocytes in whole liver lobules, while many hepatocytes were heavily labelled with BrdU. At 72 h, GJs reappeared, firstly in perinecrotic areas. At 96 h after treatment, when the injured areas had disappeared and restorative proliferation ceased, GJs were distributed evenly throughout the lobules. Immunohistochemical observation of GJs in centrilobular, perinecrotic and periportal areas after TAA-induced hepatic necrosis was confirmed by counting the number of connexin-32-positive spots in the respective areas. TJs and DSs disappeared from necrotic cells at 24 h, but then increased between 24 and 48 h in perinecrotic areas, though the increased intensity of these junctions was more evident at 48 h. At 72 h, localization of TJs and DSs returned to normal, These results suggest that during the course of acute hepatic injury, GJs (cell-cell communication) behave differently from other intercellular junctions.

DOI PubMed

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Papers - KOJIMA Takashi