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肝細胞のタイト結合におけるoccludinの役割

村田雅樹, 小島隆, 山本敏誠, 千葉英樹, 小海康夫, 澤田典均

日本病理学会会誌 ( (一社)日本病理学会 ) 92 ( 1 ) 335 - 335 2003.04

"すきま"の細胞生物学細胞間接着装置タイト結合とヒト疾患

澤田典均, 小島隆, 飛岡弘敏, 小海康夫, 千葉英樹

札幌医学雑誌 ( 札幌医科大学 ) 72 ( 1～2 ) 1 - 7 2003.04

ストレス応答MAPキナーゼ経路を介した肝細胞のギャップ及びタイト結合の発現調節機構

小島隆, 山本敏誠, 村田雅樹, 神原由季, 小海康夫, 千葉英樹, 澤田典均

日本病理学会会誌 ( (一社)日本病理学会 ) 92 ( 1 ) 336 - 336 2003.04

Epigenetic inactivation of CHFR and sensitivity to microtubule inhibitors in gastric cancer.

Cancer Res 63 8606 - 13 2003

TGF-beta/Smadsシグナルによる肝細胞のギャップ及びタイト結合の変化

小島隆, 山本敏誠, 小海康夫, 千葉英樹, 澤田典均

日本癌学会総会記事 ( (一社)日本癌学会 ) 61回 135 - 135 2002.10

IL-1βは肝細胞のギャップ及びタイト結合の発現を相反的に調節している

山本敏誠, 小島隆, 小海康夫, 千葉英樹, 澤田典均

日本癌学会総会記事 ( (一社)日本癌学会 ) 61回 134 - 134 2002.10

Cx32 formation and/or Cx32-mediated intercellular communication induces expression and function of tight junctions in hepatocytic cell line

T Kojima, DC Spray, Y Kokai, H Chiba, Y Mochizuki, N Sawada

EXPERIMENTAL CELL RESEARCH ( ACADEMIC PRESS INC ELSEVIER SCIENCE ) 276 ( 1 ) 40 - 51 2002.05

Gap junctional intercellular communication (GJIC) is thought to play a crucial role in cell differentiation. Small gap junction plaques are frequently associated with tight junction strands in hepatocytes, suggesting that gap junctions may be closely related to the role of tight junctions in the establishment of cell polarity. To examine the exact role of gap junctions in regulating tight junctions, we transfected connexin 32 (Cx32), Cx26, or Cx43 cDNAs into immortalized mouse hepatocytes derived from Cx32-deficient mice and examined the expression and function of the endogenous tight junction molecules. In transient wild-type Cx32 transfectants, immunocytochemistry revealed that endogenous occludin was in part localized at cell borders, where it was colocalized with Cx32, whereas neither was detected in parental cells. In Cx32 null hepatocytes transfected with Cx32 truncated at position 220 (R220stop), wild-type Cx26, or wild-type Cx43 cl)NAs, occludin was not detected at cell borders. In stable wild-type Cx32 transfectants, occludin, claudin-1, and ZO-1 mRNAs and proteins were significantly increased compared to parental cells and all of the proteins were colocalized with Cx32 at cell borders. Treatment with a GJIC blocker, 18beta-glycyrrhetinic acid, resulted in decreases of occludin and claudin-1 at cell borders in the stable transfectants. The induction of tight junction proteins in the stable transfectants was accompanied by an increase in both fence and barrier functions of tight junctions. Furthermore, in the stable transfectants, circumferencial actin filaments were also increased without a change of actin protein. These results indicate that Cx32 formation and/or Cx32-mediated intercellular communication may participate in the formation of functional tight junctions and actin organization. (C) 2002 Elsevier Science (USA).

DOI PubMed CiNii

Hepatocyte nuclear factor (HNF)-4 alpha induces expression of endothelial Fas ligand (FasL) to prevent cancer cell transmigration: A novel defense mechanism of endothelium against cancer metastasis

M Osanai, H Chiba, T Kojima, M Fujibe, K Kuwahara, H Kimura, M Satoh, N Sawada

JAPANESE JOURNAL OF CANCER RESEARCH ( BUSINESS CENTER ACADEMIC SOCIETIES JAPAN ) 93 ( 5 ) 532 - 541 2002.05

Endothelial Fas ligand (FasL) contributes to the "immune privilege" of tissues such as testis and eye, in which apoptosis is induced in infiltrating Fas-positive activated T cells and results in the inhibition of leukocyte extravasation. In this study, we examined the role of endothelial FasL in controlling cancer cell transmigration using rat lung endothelial (RLE) cell line bearing a doxycycline-inducible hepatocyte nuclear factor (HNF)-4alpha expression system. We showed that a detectable level of FasL was expressed in RLE cells and that this expression was markedly up-regulated and well correlated to the degree of HNF-4alpha expression in a time-dependent manner. When various cancer cells were overlaid on an RLE monolayer sheet, we examined the ability of endothelial FasL to induce massive apoptosis in Fas-expressing cancer cells and found a causal link to inhibition of the transmigration. Finally we showed that FasL was expressed in capillaries of the rat brain by immunohistochemical staining, suggesting that FasL serves its functions not only in vitro, but also in vivo. These results raise the possibility that HNF-4alpha is involved in regulating cancer cell transmigration by modulating the Fas-FasL system.

p73 is expressed in human thymic epithelial cells

S Ichimiya, T Kojima, H Momota, N Kondo, T Ozaki, A Nakagawara, ML Toribio, M Imamura, N Sato

JOURNAL OF HISTOCHEMISTRY & CYTOCHEMISTRY ( HISTOCHEMICAL SOC INC ) 50 ( 4 ) 455 - 462 2002.04

The thymus is a heterogeneous immune organ in which immature T-cells develop and eventually specialize to make certain immune responses of their own. Among various types of stromal cells in the thymus, thymic epithelial cells (TECs) have a crucially important function for presenting self-antigens and secreting cytokines to thymocytes for their maturation into T-cells. In this study we show that the p73 gene, a homologue of the tumor suppressor gene p53, was expressed in the nucleus of the human TEC in vivo and in TEC lines in vitro. Because p73 has the capacity to be a transactivator like p53, it may contribute to T-cell development in the context of TEC biology as regulated in the cell cycle and apoptosis.

The p53 family member genes are involved in the notch signal pathway

Y Sasaki, S Ishida, Morimoto, I, T Yamashita, T Kojima, C Kihara, T Tanaka, K Imai, Y Nakamura, T Tokino

JOURNAL OF BIOLOGICAL CHEMISTRY ( AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC ) 277 ( 1 ) 719 - 724 2002.01

The p53 tumor suppressor is a transcription factor that regulates cell growth and death in response to environmental stimuli such as DNA damage. p63/p51 and p73 were recently identified as members of the p53 gene family. In contrast to p53 however, p63 and p73 are rarely mutated in human cancers. Mice that lack p53 are developmentally normal, while p63 and p73 appear to play critical roles in normal development. To determine how p63 and p73 are involved in normal development, we attempted to identify target genes that are specifically regulated by p63 and/or p73 but not by p53. We found that the Jagged1 (JAG1) and Jagged2 (JAG2) genes, encoding ligands for the Notch receptors, are up-regulated by p63 and p73. Furthermore, we identified a p63-binding site in the second intron of the JAG1 gene, which can directly interact with the p63 protein in vivo, as assessed by a chromatin immunoprecipitation assay. A heterologous reporter assay revealed that this p63-binding site is a functional response element and is specific for p63. We also found a target of Notch signaling, HES-1 was up-regulated in Jurkat cells, in which Notch1 is highly expressed, when co-cultured with p63-transfected cells, suggesting that p63 can trigger the Notch signal pathway in neighboring cells. Our findings show an association between the p53 family genes and Notch signaling and suggest a potential molecular mechanism for the involvement of the p53 family genes in normal development.

DOI PubMed CiNii

Growth-suppressive function of human connexin32 in a conditional immortalized mouse hepatocyte cell line

T Kojima, M Srinivas, A Fort, M Urban, GH Lee, N Sawada, DC Spray

IN VITRO CELLULAR & DEVELOPMENTAL BIOLOGY-ANIMAL ( SOC IN VITRO BIOLOGY ) 37 ( 9 ) 589 - 598 2001.10

Mouse hepatocytes immortalized with a temperature-sensitive allele of the SV40 large T-antigen (CHST8 cells) were found to lack the high expression of the gap junction proteins Gx26 and Cx32 that characterizes normal mouse hepatocytes. expressing instead Cx43 and Cx45 at minimal levels. In order to examine the growth suppressive function of Cx32 on hepatocytes we transfected these CHST8 cells with human Cx32 complementary deoxyribonucleic acid and measured the growth rates at 33, 37, and 39 degrees C. Expression of human Cx32 and its messenger ribonucleic acid in the stable cell lines was confirmed by immunocytochemistry and by Western and Northern blots analyses. Dye transfer following lucifer yellow injection into the transfectants was extensive; Cx32 channels displayed unitary conductances of about 70 pS and were moderately voltage sensitive. When cultured at 33 and 39 degrees C, growth rates of both parental cells and transfectants were of the same level. When examined at 37 degrees C. growth rate of the transfectant, which highly expressed Cx32 at the membranes, was significantly decreased compared to the parental cells. However. no changes in the expression of Cx32 protein in the transfectants were observed between 33 and 37 degrees C. These results suggest that Cx32 expression could inhibit hepatocyte growth in vitro using the conditional immortalized cells. Cx32 transfectants using a conditional immortalized mouse hepatocyte may be useful for examining the mechanisms of growth and differentiation in hepatocytes by gap junction expression.

DOI

Global ischemia-induced increases in the gap junctional proteins connexin 32 (Cx32) and Cx36 in hippocampus and enhanced vulnerability of Cx32 knock-out mice

K Oguro, T Jover, H Tanaka, Y Lin, T Kojima, N Oguro, SY Grooms, MVL Bennett, RS Zukin

JOURNAL OF NEUROSCIENCE ( SOC NEUROSCIENCE ) 21 ( 19 ) 7534 - 7542 2001.10

Gap junctions are conductive channels that connect the interiors of coupled cells. In the hippocampus, GABA-containing hippocampal interneurons are interconnected by gap junctions, which mediate electrical coupling and synchronous firing and thereby promote inhibitory transmission. The present study was undertaken to examine the hypothesis that the gap junctional proteins connexin 32 (Cx32; expressed by oligodendrocytes, interneurons, or both), Cx36 (expressed by interneurons), and Cx43 (expressed by astrocytes) play a role in defining cell-specific patterns of neuronal death in hippocampus after global ischemia in mice. Global ischemia did not significantly alter Cx32 and Cx36 mRNA expression and slightly increased Cx43 mRNA expression in the vulnerable CA1, as assessed by Northern blot analysis and in situ hybridization. Global ischemia induced a selective increase in Cx32 and Cx36 but not Cx43 protein abundance in CA1 before onset of neuronal death, as assessed by Western blot analysis. The increase in Cx32 and Cx36 expression was intense and specific to parvalbumin-positive inhibitory interneurons of CA1, as assessed by double immunofluorescence. Protein abundance was unchanged in CA3 and dentate gyrus. The finding of increase in connexin protein without increase in mRNA suggests regulation of Cx32 and Cx36 expression at the translational or post-translational level. Cx32(Y/-) null mice exhibited enhanced vulnerability to brief ischemic insults, consistent with a role for Cx32 gap junctions in neuronal survival. These findings suggest that Cx32 and Cx36 gap junctions may contribute to the survival and resistance of GABAergic interneurons, thereby defining cell-specific patterns of global ischemia-induced neuronal death.

Gap junction expression and cell proliferation in differentiating cultures of Cx43 KO mouse hepatocytes

T Kojima, A Fort, MY Tao, M Yamamoto, DC Spray

AMERICAN JOURNAL OF PHYSIOLOGY-GASTROINTESTINAL AND LIVER PHYSIOLOGY ( AMER PHYSIOLOGICAL SOC ) 281 ( 4 ) G1004 - G1013 2001.10

Primary cultures of adult mouse hepatocytes are shown here to reexpress differentiated hepatocyte features following treatment with 2% DMSO and 10(-7) M glucagon. To examine the roles of gap junctional communication during hepatocyte growth and differentiation, we have compared treated and untreated hepatocytes from connexin (Cx) 32-deficient [Cx32 knockout (KO)] and wild-type mice. In untreated cultures, DNA replication of Cx32 KO hepatocytes was markedly higher than of wild types. Although Cx26 mRNA levels remained high at all time points in wild-type and Cx32 KO hepatocytes, Cx32 mRNA and protein in wild-type hepatocytes underwent a marked decline, which recovered in 10-day treated cultures. Increased levels of Cx26 protein and junctional conductance were observed in Cx32 KO hepatocytes at 96 h in culture, a time when cell growth rate was high. Treatment with DMSO/glucagon highly reinduced Cx26 expression in Cx32 KO hepatocytes, and such treatment reinduced expression of both Cx32 and Cx26 expression in wild types. Dye transfer was not observed following Lucifer yellow injection into DMSO/glucagon-treated Cx32 KO hepatocytes, whereas the spread was extensive in wild types. Nevertheless, high junctional conductance values were observed in treated cells from both genotypes. These studies provide a method by which the differentiated phenotype can be obtained in cultured mouse hepatocytes and provide in vitro evidence that expression of gap junctions formed of Cx32 are involved in the regulation of growth of mouse hepatocytes.

肝細胞のギャップ及びタイト結合の発現調節におけるp38MAP-kinase pathwayの関与

小島隆, 小海康夫, 千葉英樹, 澤田典均

日本癌学会総会記事 ( (一社)日本癌学会 ) 60回 93 - 93 2001.09

初代培養ラット肝細胞のギャップ及びタイト結合の発現調節に対するMAP kinase及びIP3 kinaseの抑制物質の影響

小島隆, 小海康夫, 千葉英樹, 桑原和英, 澤田典均

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 ) 54回 94 - 94 2001.05

Occludin and claudin-1 concentrate in the midbody of immortalized mouse hepatocytes during cell division

T Kojima, Y Kokai, H Chiba, M Osanai, K Kuwahara, M Mori, Y Mochizuki, N Sawada

JOURNAL OF HISTOCHEMISTRY & CYTOCHEMISTRY ( HISTOCHEMICAL SOC INC ) 49 ( 3 ) 333 - 339 2001.03

It has been believed that epithelial cells maintain tight junctions at all times, including during cell division, to provide a continuous epithelial seal. However, changes in localization of integral tight junction proteins during cell division have not been examined. In this study, using SV40-immortalized mouse hepatocytes transfected with human Cx32 cDNA, in which tight junction strands and the endogenous tight junction proteins occludin, claudin-1, ZO-1, and ZO-2 were induced, we examined changes in localization of the tight junction proteins at all stages of cell division. All tight junction proteins were present between mitotic cells and neighboring cells throughout cell division. in late telophase, the integral tight junction proteins occludin and claudin-1, but not the cytoplasmic proteins ZO-1 and ZO-2, were concentrated in the midbody between the daughter cells and were observed at cell borders between the daugher and neighboring cells. These results indicate that the integral tight junction proteins are regulated in a different manner from the cytoplasmic proteins ZO-1 and ZO-2 during cytokinesis.

Cx32 but not Cx26 is associated with tight junctions in primary cultures of rat hepatocytes

T Kojima, Y Kokai, H Chiba, M Yamamoto, Y Mochizuki, N Sawada

EXPERIMENTAL CELL RESEARCH ( ACADEMIC PRESS INC ) 263 ( 2 ) 193 - 201 2001.02

On freeze-fracture replicas, gap junctions are frequently colocalized with tight junctions. In this study, to elucidate the relationship between gap- and tight-junction proteins, we investigated the localization of gap-junction proteins Cx32 and Cx26 and tight-junction proteins occludin, claudin-1, ZO-1, and ZO-2 in primary cultured rat hepatocytes, using confocal laser microscopy. In hepatocytes cultured in 2% DMSO and 10(-7) M glucagon medium, Cx32- but not Cx26-immunoreactive lines were observed on the most sukapical plasma membrane at cell borders, while on the basolateral membrane both Cx32- and Cx26-positive spots were colocalized. Occludin-, claudin-1-, ZO-1-, and ZO-2-immunoreactive lines were also linearly observed on the most subapical plasma membrane and were colocalized with only Cx32-immunoreactive lines. In freeze-fracture analysis, many small gap-junction plaques were observed within a well-developed tight-junction strand network. The fence function of tight junctions in the cells, as examined by diffusion of labeled sphingomyelin, was well maintained. We also carried out Western blotting for Cx32 following immunoprecipitation with anti-occludin, anti-claudin-1, or anti-ZO-1 antibodies. Cx32 was detectable in all immunoprecipitates. These results suggest that Cx32 gap junctions, but not those with Cx26, are closely coordinated with the expression and function of tight junctions in hepatocytes and that Cx32 gap-junction formation may affect cell polarity through modification of tight-junction expression. (C) 2001 Academic Press.

DOI PubMed CiNii

[Development of hepatocellular carcinoma during chronic hepatitis]

Nippon Rinsho. 59 89 - 94 2001

Occludin regulates actin cytoskeleton in endothelial cells.

Cell Struct Funct 26 ( 2 ) 109 - 116 2001

DOI PubMed CiNii

Gap and tight junctions in liver: composition, regulation and function

In The Liver: Biology and Pathobiology, 4th ed 2001

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