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Occludin expression inhibits tumorigenicity and metastasis

M Osanai, N Nishikiori, M Murata, H Chiba, T Kojima, N Sawada

FASEB JOURNAL ( FEDERATION AMER SOC EXP BIOL ) 20 ( 4 ) A223 - A223 2006年03月

研究発表ペーパー・要旨（国際会議）

Glial cell line-derived neurotrophic factor (GDNF) regulates the vascular permeability of the blood-retinal barrier (BRB)

N Nishikiori, M Osanai, H Miyajima, H Chiba, T Kojima, N Sawada

FASEB JOURNAL ( FEDERATION AMER SOC EXP BIOL ) 20 ( 4 ) A636 - A637 2006年03月

研究発表ペーパー・要旨（国際会議）

The significance of interferon-gamma-triggered internalization of tight-junction proteins in inflammatory bowel disease.

Hideki Chiba, Takashi Kojima, Makoto Osanai, Norimasa Sawada

Science's STKE : signal transduction knowledge environment 2006 ( 316 ) pe1 2006年

書評論文，書評，文献紹介等

Disruption of the epithelial barrier function of tight junctions by the proinflammatory cytokine interferon (IFN)-gamma plays a fundamental role in the pathogenesis of inflammatory bowel disease and other gastrointestinal disorders, but its precise mechanism has not been established. This Perspective provides an overview of how IFN-gamma triggers barrier dysfunction and bacterial translocation in intestinal epithelial cells, and highlights the roles of internalization of tight-junction transmembrane proteins, as well as rearrangements of the cortical actin cytoskeleton, in intestinal inflammation.

DOI PubMed

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年10月

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 PubMed CiNii

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

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

EXPERIMENTAL CELL RESEARCH ( ELSEVIER INC ) 310 ( 1 ) 140 - 151 2005年10月

The tight junction (TJ) regulates epithelial cell polarity and barrier including permeability of the paracellular pathway. Occludin was the first integral membrane protein to be discovered, but it is not indispensable for the formation of TJ strands. The physiological function of occludin is still unclear, although occludin-deficient mice show very complex abnormalities in various organs without overt dysfunction of the TJ. To investigate the role of occludin in TJ expression and apoptosis regulated by survival signal transduction pathways such as MAPK and Akt, we performed primary culture of hepatocytes and established hepatic cell lines from occludin-deficient mice. In primary cultures of occludin-deficient mouse hepatocytes, claudin-2 expression and apoptosis were induced by down-regulation of the activation of MAPK and Akt. In them hepatic cell lines derived from occludin-deficient mice, claudin-2 expression and serum-free induced apoptosis were also increased by down-regulation of the activation of MAPK and Akt. Furthermore, in the hepatic cell lines transiently transfected with mouse and rat occludin genes, induction of claudin-2 expression and the apoptosis were inhibited with increases in activation of MAPK and Akt. These findings show that occludin plays a crucial role in claudin-2-dependent TJ function and the apoptosis involving MAPK and Akt signaling pathways in hepatocytes. (c) 2005 Elsevier Inc. All rights reserved.

DOI PubMed CiNii

HNF-4a provokes phosphorylation of CRB3 and develops junctional complexes and epithelial cell polarity

Hideki Chiba, Seiro Satohisa, Makoto Osanai, Shigeo Ohno, Takashi Kojima, Norimasa Sawada

CELL STRUCTURE AND FUNCTION ( JAPAN SOC CELL BIOLOGY ) 30 87 - 87 2005年06月

研究発表ペーパー・要旨（国際会議）

Glyceraldehyde-derived advanced glycation end-products preferentially induce VEGF expression and reduce GDNF expression in human astrocytes

H Miyajima, M Osanai, H Chiba, N Nishikiori, T Kojima, K Ohtsuka, N Sawada

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ( ACADEMIC PRESS INC ELSEVIER SCIENCE ) 330 ( 2 ) 361 - 366 2005年05月

The blood-brain barrier (BBB) is a biological unit composed of capillary endothelial cells and astrocytes. Here we examined the effects of various types of advanced glycation end-products (AGEs) on astrocytes and BBB-forming endothelial cells. While no type of AGE we examined changed the permeability of endothelial sheets, glyceraldehyde-derived AGE induced VEGF expression most significantly in astrocytes. The expression of glial cell line-derived neurotrophic factor (GDNF), which reduces the vascular permeability, was decreased in the astrocytes by treatment with glyceraldehyde-derived AGE. These results indicate that glyceraldehyde-derived AGE is the biologically active substance for astrocytes by regulating the VEGF and GDNF expression, which is causally contributing to an increase in the permeability of the BBB. (c) 2005 Elsevier Inc. All rights reserved.

DOI PubMed CiNii

Expression of the retinoic acid-metabolizing enzyme CYP26A1 limits programmed cell death

M Osanai, M Petkovich

MOLECULAR PHARMACOLOGY ( AMER SOC PHARMACOLOGY EXPERIMENTAL THERAPEUTICS ) 67 ( 5 ) 1808 - 1817 2005年05月

Vitamin A deficiency has been associated with increased incidence of certain types of cancer; however, the mechanisms by which vitamin A depletion promotes tumorigenesis are poorly understood. In addition all-trans-retinoic acid (RA), the most active form of vitamin A metabolites, has been shown to limit carcinogenesis in animal models and to trigger programmed cell death (apoptosis) in certain types of tumor cells. On the other hand, we show here that various cell lines overexpressing CYP26A1, a cytochrome P450 enzyme specifically involved in the catabolic inactivation of RA, exhibit increased resistance to various apoptogenic factors, including death receptor ligands such as tumor necrosis factor-related apoptosis-inducing ligand. This resistance could be reversed by pretreatment with ketoconazole, a broad-spectrum inhibitor of cytochrome P450 enzymes. In addition, synthetic retinoids Am80 (4[( 5,6,7,8-tetrahydro5,5,8,8- tetramethyl-2-naphthalenyl) carbamoyl] benzoic acid) and Am580 [ 4( 5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphtamido) benzoic acid], which are resistant to CYP26A1 metabolism, can restore the sensitivity of these cells to apoptogens. Thus, these findings support the idea that CYP26 expression levels may play a role in determining cellular commitment to apoptosis, and increased RA metabolism may be at least partially responsible for these observed effects.

DOI

Expression of the retinoic acid-metabolizing enzyme CYP26A1 limits programmed cell death

M Osanai, M Petkovich

MOLECULAR PHARMACOLOGY 67 ( 5 ) 1808 - 1817 2005年05月

DOI

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

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

EXPERIMENTAL CELL RESEARCH ( ELSEVIER INC ) 312 ( 2 ) 111 - 120 2005年01月

The apical microvilli are closely related with the development and the maintenance of cell polarization, and the length of microvilli varies in a regular way among cell types. Ezrin, a member of the ezrin/radixin/moesin (ERM) family, seems to be involved in the formation and stabilization of the apical microvilli. We found that phosphorylation of ezrin caused elongation of microvilli via a p38 MAP-kinase signaling pathway in all immortalized mouse hepatic cell line. When, in the oncogenic Raf-1-transfected mouse hepatic cell line, epithelial to mesenchymal transition (EMT) indicated as down-regulation of E-cadherin and up-regulation of Snail occurred, loss of microvilli and down-regulation of ezrin hot not radixin and moesin were also observed. In the Raf-1 transfectants treated with the MAP-kinase inhibitor PD98059 and the p38 MAP-kinase inhibitor SB203580, the numbers of microvilli and the expression of ezrin, E-cadherin and Snail were recovered. More interestingly, treatment with SB203580 induced elongation of microvilli and increased phosphorylation of ezrin (at Thr-567 and Tyr-353). Phosphorylated ezrin-positive clots were colocalized with actin-positive dots on the surface of some Raf-1 transfectants treated with SB203580. These results suggested that phosphorylation of ezrin via the p38 MAP-kinase signaling pathway might be involved in the formation of microvilli during development of epithelial cell polarization. (c) 2005 Elsevier Inc. All rights reserved.

DOI PubMed CiNii

Activation of p21 (CIP1/WAF1) gene expression and inhibition of cell proliferation by overexpression of hepatocyte nuclear factor-4 alpha

H Chiba, T Itoh, S Satohisa, N Sakai, H Noguchi, M Osanai, T Kojima, N Sawada

EXPERIMENTAL CELL RESEARCH ( ELSEVIER INC ) 302 ( 1 ) 11 - 21 2005年01月

The F9 murine embryonal carcinoma cell line provides an attractive system for studying epithelial differentiation and antiproliferative processes. We have recently established F9 cells expressing doxycycline-inducible hepatocyte nuclear factor (HNF)-4alpha and shown that HNF-4alpha triggers the gene expression of tight-junction molecules, occludin, claudin-6, and claudin-7, as well as formation of functional tight junctions and polarized epithelial morphology (Exp. Cell Res. 286, [2003] 288). Since these events were very similar to those induced by retinoids, we investigated whether HNF-4alpha, like retinoid receptors, was involved in the control of cell proliferation. We herein show that HNF-4alpha up-regulates expression of the p21 gene, but not the p15, p16, p18, p19, or p27 gene, in a p53-independent manner, and inhibits cell growth in F9 cells. Similar results were observed in rat lung endothelial cells, in which expression of HNF-4alpha is conditionally induced by doxycycline. Furthermore, we demonstrate, by reporter assay, that HNF-4alpha significantly elevates the transcriptional activity of the p21 promoter. Since, HNF-4alpha is expressed not only in the liver but also in organs containing epithelial cells, such as kidney, intestine, pancreas, and stomach, it might also play critical roles in the regulation of epithelial morphogenesis and proliferation in these organs. (C) 2004 Elsevier Inc, All rights reserved.

DOI PubMed CiNii

Oncogenic Raf-1 regulates epithelial to mesenchymal transition via distinct signal transduction pathways in an immortalized mouse hepatic cell line

MD Lan, T Kojima, M Osanai, H Chiba, N Sawada

CARCINOGENESIS ( OXFORD UNIV PRESS ) 25 ( 12 ) 2385 - 2395 2004年12月

The epithelial to mesenchymal transition (EMT) is considered to be an important event during malignant tumor progression and metastasis. Although Raf/MEK/ERK signaling causes EMT, the mechanisms, including the signaling pathways, are as yet unclear. In the present study we have examined the effects of signal transduction pathways on oncogenic Raf-1-induced EMT, using an immortalized mouse hepatic cell line. Oncogenic Raf-1-induced EMT is characterized by down-regulation of adherens and tight junctions and the reorganization of actin. An active Raf-1 gene was introduced into a mouse hepatic cell line which was then treated with the MAP kinase inhibitor PD98059, the p38 MAP kinase inhibitor SB203580, the PI3 kinase inhibitor LY294002 or the c-Src tyrosine kinase inhibitor PP2. The expression and localization of the adherens and tight junction proteins E-cadherin, occludin, ZO-1, claudin-1 and claudin-2 were determined by western blotting, RT-PCR and immunocytochemistry. The barrier function of tight junctions was assessed by measurements of transepithelial electric resistance (TER) and permeability in terms of fluxes of [C-14]mannitol and [C-14]inulin. In Raf-1-transfected cells expression of occludin and claudin-2 was markedly down-regulated at the protein and mRNA levels and the TER value was decreased, while the permeability was increased. The distribution of ZO-1, pancadherin and F-actin was changed from linear to zipper-like structures at cell borders. In Raf-1-transfected cells treated with PD98059 and SB203580, but not LY294002, expression and localization of claudin-2, but not occludin, recovered, together with barrier function, measured as the TER value. The distributions of ZO-1, pancadherin and F-actin also recovered on treatment with PD98059 and SB203580, but not LY294002. Expression and localization of occludin recovered slightly on treatment with PP2. Thus, oncogenic Raf-1 regulates EMT via distinct MAP kinase, p38 MAP kinase and c-Src tyrosine kinase signal pathways in the mouse hepatic cell line.

DOI

Unique cellular features of peripheral primitive neuroectodermal tumor: Ultrastructural evidence of its unique cytodifferentiation

Makoto Osanai, Jun Yamaguchi, Keisuke Kikuchi, Masaaki Satoh, Norimasa Sawada

Medical Electron Microscopy 37 ( 3 ) 193 - 197 2004年

We present a case of primitive neuroectodermal tumor (PNET) showing a unique differentiation phenotype based on ultrastructural observation. Rapidly growing tumor involving the retroperitoneum of a 68-year-old woman was characterized by histological findings including proliferation of diffuse poorly differentiated small round cells with scattered rosette formation similar to Homer-Wright type and by the ultrastructure demonstrating cytoplasmic neurosecretary granules and short cytoplasmic cilial structures. These cells revealed immunoreactivity only with neuron-specific enolase (NSE). We also observed a chromosomal translocation, t (11 22) (q24 q12), which is an identical recurrent alteration found in the neoplastic cells in the spectrum of PNETs. These findings may support the explanation that tumor cells observed in our case shared phenotypes of both neuronal and ependymal cell lineages and give a unique insight suggesting the possible histogenesis of PNETs. © The Clinical Electron Microscopy Society of Japan 2004.

DOI PubMed

Unique cellular features of peripheral primitive neuroectodermal tumor: Ultrastructural evidence of its unique cytodifferentiation

Makoto Osanai, Jun Yamaguchi, Keisuke Kikuchi, Masaaki Satoh, Norimasa Sawada

Medical Electron Microscopy 37 ( 3 ) 193 - 197 2004年

DOI PubMed

Tight junctions and human diseases

Norimasa Sawada, Masaki Murata, Keisuke Kikuchi, Makoto Osanai, Hirotoshi Tobioka, Takashi Kojima, Hideki Chiba

Medical Electron Microscopy 36 ( 3 ) 147 - 156 2003年09月

書評論文，書評，文献紹介等

Tight junctions are intercellular junctions adjacent to the apical end of the lateral membrane surface. They have two functions, the barrier (or gate) function and the fence function. The barrier function of tight junctions regulates the passage of ions, water, and various macromolecules, even of cancer cells, through paracellular spaces. The barrier function is thus relevant to edema, jaundice, diarrhea, and blood-borne metastasis. On the other hand, the fence function maintains cell polarity. In other words, tight junctions work as a fence to prevent intermixing of molecules in the apical membrane with those in the lateral membrane. This function is deeply involved in cancer cell biology, in terms of loss of cell polarity. Of the proteins comprising tight junctions, integral membrane proteins occludin, claudins, and JAMs have been recently discovered. Of these molecules, claudins are exclusively responsible for the formation of tight-junction strands and are connected with the actin cytoskeleton mediated by ZO-1. Thus, both functions of tight junctions are dependent on the integrity of the actin cytoskeleton as well as ATP. Mutations in the claudin14 and the claudin16 genes result in hereditary deafness and hereditary hypomagnesemia, respectively. Some pathogenic bacteria and viruses target and affect the tight-junction function, leading to diseases. In this review, the relationship between tight junctions and human diseases is summarized.

DOI PubMed CiNii

Hepatocyte nuclear factor (HNF)-4 alpha triggers formation of functional tight junctions and establishment of polarized epithelial morphology in F9 embryonal carcinoma cells

H Chiba, T Gotoh, T Kojima, S Satohisa, K Kikuchi, M Osanai, N Sawada

EXPERIMENTAL CELL RESEARCH ( ACADEMIC PRESS INC ELSEVIER SCIENCE ) 286 ( 2 ) 288 - 297 2003年06月

F9 murine embryonal carcinoma cells provide an attractive system for facilitating molecular mechanisms for epithelial morphogenesis, since they have the capability of differentiating into polarized epithelial cells bearing an apical junctional complexes. We previously showed that a specific retinoid X receptor-retinoic acid receptor heterodimer transduced retinoid signals for biogenesis of functional tight junctions in F9 cells (Exp. Cell Res. 263, (2001) 163). In the present study we generated F9 cells expressing doxycycline-inducible hepatocyte nuclear factor (HNF)-4alpha, a nuclear receptor. We herein show that induction of HNF-4alpha initiates differentiation of F9 cells to polarized epithelial cells, in which tight-junction proteins occludin, claudin-6, claudin-7, and ZO-1 are concentrated at the apical-most regions of lateral membranes. Expression of occludin, claudin-6, and claudin-7 was induced in the cells by doxycycline treatment in a dose- and time-dependent manner, in terms of the amount of HNF-4a. In contrast, expression levels of ZO-1, ZO-2, E-cadherin, and P-catenin were not altered by HNF-4a. We also demonstrate, by analysis of diffusion of labeled sphingomyelin, that the fence function of tight junctions is achieved by induction of HNF-4a. These findings indicate that HNF-4a triggers de novo formation of functional tight junctions and establishment of epithelial cell polarity. (C) 2003 Elsevier Science (USA). All rights reserved.

DOI PubMed CiNii

Functions of hepatocyte nuclear factor-4 alpha in epithelial differentiation and proliferation of F9 embryonal carcinoma cells

H Chiba, T Goto, T Itoh, K Kikuchi, M Osanai, T Kojima, N Sawada

MOLECULAR BIOLOGY OF THE CELL ( AMER SOC CELL BIOLOGY ) 13 494A - 495A 2002年11月

研究発表ペーパー・要旨（国際会議）

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年05月

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.

Transient increase in telomerase activity of proliferating fibroblasts and endothelial cells in granulation tissue of the human skin

M Osanai, T Tamaki, M Yonekawa, A Kawamura, N Sawada

WOUND REPAIR AND REGENERATION ( BLACKWELL PUBLISHING INC ) 10 ( 1 ) 59 - 66 2002年01月

Although granulation tissue formation is an important step for second-intention wound healing, the molecular events underlying this process are still poorly understood, To investigate the role of telomerose in the formation of granulation tissue, we measured the activity of this enzyme and determined the expression and localization of human telomerase reverse transcriptase mRNA using human skin samples, Telomerase activity in the tip of the granulation tissue where fibroblasts actively proliferate was detected at a level 5,6 +/- 1.5 times higher than that at the edge of the tissue when using a polymerase chain reaction-based telomeric repeat amplification protocol assay coupled with enzyme-linked immunosorbent assay, This, together with the findings from semiquantitative reverse transcriptase-polymerase chain reaction and in situ hybridization of human telomerase reverse transcriptase, revealed that proliferating cell nuclear antigen-positive fibroblasts and endothelial cells in the progressing granulation tissue showed de novo activation of telomerase with high human telomerase reverse transcriptase mRNA expression, This condition may be a prolongation of cellular replicative capacity taking advantage of the positive regulatory dynamics of cell growth, We conclude that the regulation of telomerase activity may play an important role in granulation tissue formation in wound healing.

DOI

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

Osanai M, Chiba H, Kojima T, Fujibe M, Satoh M, Sawada N

Jpn J Cancer Res. 93 ( 5 ) 532 - 541 2002年

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)-4α 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-4α 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-4α is involved in regulating cancer cell transmigration by modulating the Fas-FasL system.

DOI PubMed

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