The roles of tight junction protein cingulin in human endometrioid endometrial cancer.

The bicellular tight junction molecule cingulin (CGN) binds to microtubules in centrosomes. Furthermore, CGN contributes to the tricellular tight junction (tTJ) proteins lipolysis-stimulated lipoprotein receptor (LSR) and tricellulin (TRIC). CGN as well as LSR decreased during the malignancy of endometrioid endometrial cancer (EEC). Although tTJ protein LSR is involved in the malignancy of some cancers, including EEC, the role of CGN is unknown. In this study, we investigated the roles of CGN with tTJ proteins in human EEC cells by using the CGN-overexpressing EEC cell line Sawano. In 2D cultures, CGN was colocalized with LSR and TRIC at tTJ or at γ-tubulin-positive centrosomes. In immunoprecipitation with CGN antibodies, CGN directly bound to LSR, TRIC, and ß-tubulin. Knockdown of CGN by the siRNA decreased the epithelial barrier and enhanced cell proliferation, migration and invasion, as well as knockdown of LSR. In the Sawano cells cocultured with normal human endometrial stromal cells, knockdown of CGN decreased expression of LSR and TRIC via MAPK and AMPK pathways. In 2.5D cultures, knockdown of CGN induced the formation of abnormal cysts and increased the permeability of FD-4 to the lumen. In 2D and 2.5D cultures, treatment with ß-estradiol with or without EGF or TGF-ß decreased CGN expression and the epithelial permeability barrier and enhanced cell migration, and pretreatment with EW7197+AG1478, U0126 or an anti-IL-6 antibody prevented this. In conclusion, CGN, with tTJ proteins might suppress the malignancy of human EEC and its complex proteins are sensitive to estrogen and growth factors derived from stromal cells.

The Roles and Regulatory Mechanisms of Tight Junction Protein Cingulin and Transcription Factor Forkhead Box Protein O1 in Human Lung Adenocarcinoma A549 Cells and Normal Lung Epithelial Cells.

Tight junction (TJ) protein cingulin (CGN) and transcription factor forkhead box protein O1 (FOXO1) contribute to the development of various cancers. Histone deacetylase (HDAC) inhibitors have a potential therapeutic role for some cancers. HDAC inhibitors affect the expression of both CGN and FOXO1. However, the roles and regulatory mechanisms of CGN and FOXO1 are unknown in non-small cell lung cancer (NSCLC) and normal human lung epithelial (HLE) cells. In the present study, to investigate the effects of CGN and FOXO1 on the malignancy of NSCLC, we used A549 cells as human lung adenocarcinoma and primary human lung epithelial (HLE) cells as normal lung tissues and performed the knockdown of CGN and FOXO1 by siRNAs. Furthermore, to investigate the detailed mechanisms in the antitumor effects of HDAC inhibitors for NSCLC via CGN and FOXO1, A549 cells and HLE cells were treated with the HDAC inhibitors trichostatin A (TSA) and Quisinostat (JNJ-2648158). In A549 cells, the knockdown of CGN increased bicellular TJ protein claudin-2 (CLDN-2) via mitogen-activated protein kinase/adenosine monophosphate-activated protein kinase (MAPK/AMPK) pathways and induced cell migration, while the knockdown of FOXO1 increased claudin-4 (CLDN-4), decreased CGN, and induced cell proliferation. The knockdown of CGN and FOXO1 induced cell metabolism in A549 cells. TSA and Quisinostat increased CGN and tricellular TJ protein angulin-1/lipolysis-stimulated lipoprotein receptor (LSR) in A549. In normal HLE cells, the knockdown of CGN and FOXO1 increased CLDN-4, while HDAC inhibitors increased CGN and CLDN-4. In conclusion, the knockdown of CGN via FOXO1 contributes to the malignancy of NSCLC. Both HDAC inhibitors, TSA and Quisinostat, may have potential for use in therapy for lung adenocarcinoma via changes in the expression of CGN and FOXO1.

The interplay between the epithelial permeability barrier, cell migration and mitochondrial metabolism of growth factors and their inhibitors in a human endometrial carcinoma cell line.

It is known that there are abnormalities of tight junction functions, cell migration and mitochondrial metabolism in human endometriosis and endometrial carcinoma. In this study, we investigated the effects of growth factors and their inhibitors on the epithelial permeability barrier, cell migration and mitochondrial metabolism in 2D and 2.5D cultures of human endometrioid endometrial carcinoma Sawano cells. We also investigated the changes of bicellular and tricellular tight junction molecules and ciliogenesis induced by these inhibitors. The growth factors TGF-ß and EGF affected the epithelial permeability barrier, cell migration and expression of bicellular and tricellular tight junction molecules in 2D and 2.5D cultures of Sawano cells. EW-7197 (a TGF-ß receptor inhibitor), AG1478 (an EGFR inhibitor) and SP600125 (a JNK inhibitor) affected the epithelial permeability barrier, cell migration and mitochondrial metabolism and prevented the changes induced by TGF-ß and EGF in 2D and 2.5D cultures. EW-7197 and AG1478 induced ciliogenesis in 2.5D cultures. In conclusion, TGF-ß and EGF promoted the malignancy of endometrial cancer via interplay among the epithelial permeability barrier, cell migration and mitochondrial metabolism. EW-7197 and AG1478 may be useful as novel therapeutic treatments options for endometrial cancer.

Downregulation of angulin-1/LSR induces malignancy via upregulation of EGF-dependent claudin-2 and TGF-ß-dependent cell metabolism in human lung adenocarcinoma A549 cells.

Abnormal expression of bicellular tight junction claudins, including claudin-2 are observed during carcinogenesis in human lung adenocarcinoma. However, little is known about the role of tricellular tight junction molecule angulin-1/lipolysis-stimulated lipoprotein receptor (LSR). In the lung adenocarcinoma tissues examined in the present study, expression of claudin-2 was higher than in normal lung tissues, while angulin-1/LSR was poorly or faintly expressed. We investigated how loss of angulin-1/LSR affects the malignancy of lung adenocarcinoma cell line A549 and normal human lung epithelial (HLE) cells. The EGF receptor tyrosine kinase inhibitor AG1478 prevented the increase of claudin-2 expression induced by EGF in A549 cells. Knockdown of LSR induced expression of claudin-2 at the protein and mRNA levels and AG1478 prevented the upregulation of claudin-2 in A549 cells. Knockdown of LSR induced cell proliferation, cell migration and cell metabolism in A549 cells. Knockdown of claudin-2 inhibited the cell proliferation but did not affect the cell migration or cell metabolism of A549 cells. The TGF-ß type I receptor inhibitor EW-7197 prevented the decrease of LSR and claudin-2 induced by TGF-ß1 in A549 cells and 2D culture of normal HLE cells. EW-7197 prevented the increase of cell migration and cell metabolism induced by TGF-ß1 in A549 cells. EW-7197 prevented the increase of epithelial permeability of FITC-4kD dextran induced by TGF-ß1 in 2.5D culture of normal HLE cells. In conclusion, downregulation of angulin-1/LSR induces malignancy via EGF-dependent claudin-2 and TGF-ß-dependent cell metabolism in human lung adenocarcinoma.

Oxygen-Glucose Deprivation Decreases the Motility and Length of Axonal Mitochondria in Cultured Dorsal Root Ganglion Cells of Rats.

Controlling axonal mitochondria is important for maintaining normal function of the neural network. Oxygen-glucose deprivation (OGD), a model used for mimicking ischemia, eventually induces neuronal cell death similar to axonal degeneration. Axonal mitochondria are disrupted during OGD-induced neural degeneration; however, the mechanism underlying mitochondrial dysfunction has not been completely understood. We focused on the dynamics of mitochondria in axons exposed to OGD; we observed that the number of motile mitochondria significantly reduced in 1 h following OGD exposure. In our observation, the decreased length of stationary mitochondria was affected by the following factors: first, the halt of motile mitochondria; second, the fission of longer stationary mitochondria; and third, a transformation from tubular to spherical shape in OGD-exposed axons. Motile mitochondria reduction preceded stationary mitochondria fragmentation in OGD exposure; these conditions induced the decrease of stationary mitochondria in three different ways. Our results suggest that mitochondrial morphological changes precede the axonal degeneration while ischemia-induced neurodegeneration.

LSR antibody promotes apoptosis and disrupts epithelial barriers via signal pathways in endometrial cancer.

Lipolysis-stimulated lipoprotein receptor (LSR), a lipid metabolism-related factor localized in tricellular tight junctions (tTJs), plays an important role in maintaining the epithelial barrier. LSR is highly expressed in well-differentiated endometrial endometrioid carcinoma (EEC), and its expression decreases during malignancy. Angubindin-1, a novel LSR ligand peptide, regulates tTJs without cytotoxicity, enhances paracellular permeability, and regulates epithelial barrier via c-Jun N-terminal kinase (JNK)/cofilin. In this study, we investigated the immune-modulatory roles of an anti-LSR antibody in the treatment of EEC in vitro compared to those of angubindin-1. We prepared an antibody against the extracellular N-terminal domain of human LSR (LSR-N-ab) and angubindin-1. EEC cell-line Sawano cells in 2D and 2.5D cultures were treated with 100 µg/ml LSR-N-ab or 2.5 µg/ml angubindin-1 with or without protein tyrosine kinase 2ß inhibitor PF431396 (PF43) and JNK inhibitor SP600125 (SP60) at 10 µM. Treatment with LSR-N-ab and angubindin-1 decreased LSR at the membranes of tTJs and the activity of phosphorylated LSR and phosphorylated cofilin in 2D culture. Treatment with LSR-N-ab and angubindin-1 decreased the epithelial barrier measured as TEER values in 2D culture and enhanced the epithelial permeability of FD-4 in 2.5D culture. Treatment with LSR-N-ab, but not angubindin-1, induced apoptosis in 2D culture. Pretreatment with PF43 and SP60 prevented all the changes induced by treatment with LSR-N-ab and angubindin-1. Treatment with LSR-N-ab and angubindin-1 enhanced the cell metabolism measured as the mitochondrial respiration levels in 2D culture. LSR-N-ab and angubindin-1 may be useful for therapy of human EEC via enhanced apoptosis or drug absorption.

Atypical Macropinocytosis Contributes to Malignant Progression: A Review of Recent Evidence in Endometrioid Endometrial Cancer Cells.

Macropinocytosis is an essential mechanism for the non-specific uptake of extracellular fluids and solutes. In recent years, additional functions have been identified in macropinocytosis, such as the intracellular introduction pathway of drugs, bacterial and viral infection pathways, and nutritional supplement pathway of cancer cells. However, little is known about the changes in cell function after macropinocytosis. Recently, it has been reported that macropinocytosis is essential for endometrial cancer cells to initiate malignant progression in a dormant state. Macropinocytosis is formed by a temporary split of adjacent bicellular junctions of epithelial sheets, rather than from the apical surface or basal membrane, as a result of the transient reduction of tight junction homeostasis. This novel type of macropinocytosis has been suggested to be associated with the malignant pathology of endometriosis and endometrioid endometrial carcinoma. This review outlines the induction of malignant progression of endometrial cancer cells by macropinocytosis based on a new mechanism and the potential preventive mechanism of its malignant progression.

Inhibition of HDAC and Signal Transduction Pathways Induces Tight Junctions and Promotes Differentiation in p63-Positive Salivary Duct Adenocarcinoma.

BACKGROUND: The p53 family p63 is essential for the proliferation and differentiation of various epithelial basal cells. It is overexpressed in several cancers, including salivary gland neoplasia. Histone deacetylases (HDACs) are thought to play a crucial role in carcinogenesis, and HDAC inhibitors downregulate p63 expression in cancers. METHODS: In the present study, to investigate the roles and regulation of p63 in salivary duct adenocarcinoma (SDC), human SDC cell line A253 was transfected with siRNA-p63 or treated with the HDAC inhibitors trichostatin A (TSA) and quisinostat (JNJ-26481585). RESULTS: In a DNA array, the knockdown of p63 markedly induced mRNAs of the tight junction (TJ) proteins cingulin (CGN) and zonula occuludin-3 (ZO-3). The knockdown of p63 resulted in the recruitment of the TJ proteins, the angulin-1/lipolysis-stimulated lipoprotein receptor (LSR), occludin (OCLN), CGN, and ZO-3 at the membranes, preventing cell proliferation, and leading to increased cell metabolism. Treatment with HDAC inhibitors downregulated the expression of p63, induced TJ structures, recruited the TJ proteins, increased the epithelial barrier function, and prevented cell proliferation and migration. CONCLUSIONS: p63 is not only a diagnostic marker of salivary gland neoplasia, but it also promotes the malignancy. Inhibition of HDAC and signal transduction pathways is, therefore, useful in therapy for p63-positive SDC cells.

FOXO3/TGF-ß signal-dependent ciliogenesis and cell functions during differentiation of temperature-sensitive mouse cochlear precursor hair cells.

The transcription factor FOXO3 is necessary to preserve cochlear hair cells. Growth factors, including TGF-ß, closely contribute to cochlear hair cell regeneration. In the present study, to investigate the roles of FOXO3 in the ciliogenesis and cell functions of cochlear hair cells, UB/OC-2 temperature-sensitive mouse cochlear precursor hair cells were treated with TGF-ß receptor type 1 inhibitor EW-7197 or EGF receptor inhibitor AG-1478 after transfection with or without siRNA-FOXO3a. GeneChip analysis revealed that treatment with EW-7197 increased Foxo3 genes and decreased genes of Smads. During cell differentiation, treatment with EW-7197 or AG-1478 induced an increase in length of cilia-like structures that were positive for acetylated tubulin and inhibited cell migration. Treatment with EW-7197 also increased cell metabolism measured as mitochondrial basal respiration (oxygen consumption rate). The effects of EW-7197 were stronger than those of AG-1478. Knockdown of FOXO3 prevented the growth of cilia-like structures induced by EW-7197 or AG-1478 and induced cell migration under treatment with EW-7197. No change of the epithelial cell polarity molecule PAR3 was observed with any treatment. Treatment with the antimicrobial agent amikacin prevented the growth of cilia-like structures induced by EW-7197 and induced apoptosis. Pretreatment with the glucocorticoid dexamethasone inhibited the apoptosis induced by amikacin. This in vitro model of mouse cochlear hair cells suggests that FOXO3/TGF-ß signaling plays a crucial role in ciliogenesis and cell functions during differentiation of cochlear hair cells. This model is useful for analysis of the mechanisms of hearing loss and to find therapeutic agents to prevent it.

Dysfunction of epithelial permeability barrier induced by HMGB1 in 2.5D cultures of human epithelial cells.

Airway and intestinal epithelial permeability barriers are crucial in epithelial homeostasis. High mobility group box 1 (HMGB1), increased by various stimuli, is involved in the induction of airway inflammation, as well as the pathogenesis of inflammatory bowel disease. HMGB1 enhances epithelial hyperpermeability. Two-and-a-half dimensional (2.5D) culture assays are experimentally convenient and induce cells to form a more physiological tissue architecture than 2D culture assays for molecular transfer mechanism analysis. In 2.5D culture, treatment with HMGB1 induced permeability of FITC-dextran into the lumen formed by human lung, nasal and intestinal epithelial cells. The tricellular tight junction molecule angulin-1/LSR is responsible for the epithelial permeability barrier at tricellular contacts and contributes to various human airway and intestinal inflammatory diseases. In this review, we indicate the mechanisms including angulin-1/LSR and multiple signaling in dysfunction of the epithelial permeability barrier induced by HMGB1 in 2.5D culture of human airway and intestinal epithelial cells.

The Roles of Tricellular Tight Junction Protein Angulin-1/Lipolysis-Stimulated Lipoprotein Receptor (LSR) in Endometriosis and Endometrioid-Endometrial Carcinoma.

Tight junction proteins play roles beyond permeability barriers functions and control cell proliferation and differentiation. The relation between tight junctions and the signal transduction pathways affects cell growth, invasion and migration. Abnormality of tight junction proteins closely contributes to epithelial mesenchymal transition (EMT) and malignancy of various cancers. Angulin-1/lipolysis-stimulated lipoprotein receptor (LSR) forms tricellular contacts that has a barrier function. Downregulation of angulin-1/LSR correlates with the malignancy in various cancers, including endometrioid-endometrial carcinoma (EEC). These alterations have been shown to link to not only multiple signaling pathways such as Hippo/YAP, HDAC, AMPK, but also cell metabolism in ECC cell line Sawano. Moreover, loss of angulin-1/LSR upregulates claudin-1, and loss of apoptosis stimulating p53 protein 2 (ASPP2) downregulates angulin-1/LSR. Angulin-1/LSR and ASPP2 concentrate at both midbody and centrosome in cytokinesis. In EEC tissues, angulin-1/LSR and ASPP2 are reduced and claudin-2 is overexpressed during malignancy, while in the tissues of endometriosis changes in localization of angulin-1/LSR and claudin-2 are seen. This review highlights how downregulation of angulin-1/LSR promotes development of endometriosis and EEC and discusses about the roles of angulin-1/LSR and its related proteins, including claudins and ASPP2.

Translocation of LSR from tricellular corners causes macropinocytosis at cell-cell interface as a trigger for breaking out of contact inhibition.

Withdrawal from contact inhibition is necessary for epithelial cancer precursor cells to initiate cell growth and motility. Nevertheless, little is understood about the mechanism for the sudden initiation of cell growth under static conditions. We focused on cellular junctions as one region where breaking out of contact inhibition occurs. In well-differentiated endometrial cancer cells, Sawano, the ligand administration for tricellular tight junction protein LSR, which transiently decreased the robust junction property, caused an abrupt increase in cell motility and consequent excessive multilayered cell growth despite being under contact inhibition conditions. We observed that macropinocytosis essentially and temporarily occurred as an antecedent event for the above process at intercellular junctions without disruption of the junction apparatus but not at the apical plasma membrane. Collectively, we concluded that the formation of macropinocytosis, which is derived from tight junction-mediated signaling, was triggered for the initiation of cell growth in static precancerous epithelium.

Effects of HMGB1 on Tricellular Tight Junctions via TGF-ß Signaling in Human Nasal Epithelial Cells.

The airway epithelium of the human nasal mucosa acts as a physical barrier that protects against inhaled substances and pathogens via bicellular and tricellular tight junctions (bTJs and tTJs) including claudins, angulin-1/LSR and tricellulin. High mobility group box-1 (HMGB1) increased by TGF-ß1 is involved in the induction of nasal inflammation and injury in patients with allergic rhinitis, chronic rhinosinusitis, and eosinophilic chronic rhinosinusitis. However, the detailed mechanisms by which this occurs remain unknown. In the present study, to investigate how HMGB1 affects the barrier of normal human nasal epithelial cells, 2D and 2.5D Matrigel culture of primary cultured human nasal epithelial cells were pretreated with TGF-ß type I receptor kinase inhibitor EW-7197 before treatment with HMGB1. Knockdown of angulin-1/LSR downregulated the epithelial barrier. Treatment with EW-7197 decreased angulin-1/LSR and concentrated the expression at tTJs from bTJs and increased the epithelial barrier. Treatment with a binder to angulin-1/LSR angubindin-1 decreased angulin-1/LSR and the epithelial barrier. Treatment with HMGB1 decreased angulin-1/LSR and the epithelial barrier. In 2.5D Matrigel culture, treatment with HMGB1 induced permeability of FITC-dextran (FD-4) into the lumen. Pretreatment with EW-7197 prevented the effects of HMGB1. HMGB1 disrupted the angulin-1/LSR-dependent epithelial permeability barriers of HNECs via TGF-ß signaling in HNECs.

Effects of histone deacetylase inhibitors Tricostatin A and Quisinostat on tight junction proteins of human lung adenocarcinoma A549 cells and normal lung epithelial cells.

Histone deacetylase (HDAC) inhibitors have a potential therapeutic role for non-small cell lung cancer (NSCLC). However, more preclinical studies of HDAC inhibitors in NSCLC and normal lung epithelial cells are required to evaluate their antitumor activities and mechanisms. The bicellular tight junction molecule claudin-2 (CLDN-2) is highly expressed in lung adenocarcinoma tissues and increase the proliferation of adenocarcinoma cells. Downregulation of the tricellular tight junction molecule angulin-1/LSR induces malignancy via EGF-dependent CLDN-2 and TGF-ß-dependent cellular metabolism in human lung adenocarcinoma cells. In the present study, to investigate the detailed mechanisms of the antitumor activities of HDAC inhibitors in lung adenocarcinoma, human lung adenocarcinoma A549 cells and normal lung epithelial cells were treated with the HDAC inibitors Trichostatin A (TSA) and Quisinostat (JNJ-2648158) with or without TGF-ß. Both HDAC inhibitors increased anguin-1/LSR, decrease CLDN-2, promoted G1 arrest and prevented the migration of A549 cells. Furthermore, TSA but not Quisinostat with or without TGF-ß induced cellular metabolism indicated as the mitochondrial respiration measured using the oxygen consumption rate. In normal human lung epithelial cells, treatment with TSA and Quisinostat increased expression of LSR and CLDN-2 and decreased that of CLDN-1 with or without TGF-ß in 2D culture. Quisinostat but not TSA with TGF-ß increased CLDN-7 expression in 2D culture. Both HDAC inhibitors prevented disruption of the epithelial barrier measured as the permeability of FD-4 induced by TGF-ß in 2.5D culture. TSA and Quisinostat have potential for use in therapy for lung adenocarcinoma via changes in the expression of angulin-1/LSR and CLDN-2.

Pyk2 inhibitor prevents epithelial hyperpermeability induced by HMGB1 and inflammatory cytokines in Caco-2 cells.

The non-receptor protein tyrosine kinase 2ß (Pyk2) phosphorylated tricellular tight junction (tTJ) molecules angulin-1/LSR and tricellulin (TRIC) and the inhibitor PF-431396 (PF43) suppress angulin-1/LSR and TRIC recruitment to tTJs. The disruption of the intestinal epithelial barrier by high mobility group box 1 (HMGB1) and the inflammatory cytokines TNFα and IFNγ contributes to downregulation of angulin-1/LSR and TRIC in 2.5D culture of Caco-2 cells as a novel model of inflammatory bowel disease (IBD). In the present study, to investigate the roles of Pyk2 phosphorylated angulin-1/LSR and TRIC in the intestinal epithelial barrier, 2D and 2.5D cultures of Caco-2 cells were treated with the Pyk2 inhibitor PF-43 with or without HMGB1, inflammatory cytokines TNFα and IFNγ. Treatment with PF-43 increased expression of angulin-1/LSR, phosphorylated AMPK and phosphorylated MAPK and decreased that of phosphorylated JNK, with upregulation of the epithelial barrier and cellular metabolism measured as basal oxygen consumption rate (OCR) and ATP production in 2D culture. Treatment with PF-43 prevented the downregulation of the epithelial barrier by HMGB1 and inflammatory cytokines in 2D culture. Treatment with PF-43 prevented the epithelial hyperpermeability induced by HMGB1 and inflammatory cytokines in 2.5D culture. In 2.5D culture, treatment with PF-43 inhibited the decreases of angulin-1/LSR, TRIC, pJNK, pAMPK and pMAPK induced by HMGB1 and the inflammatory cytokines. Treatment with PF-43 inhibited in part the induced phosphorylation of the serine of angulin-1/LSR and TRIC. Pyk2 inhibitor PF-43 may have potential for use in therapy for IBD via its actions with regard to phosphorylated tTJs and cellular metabolism.

HDAC inhibitors suppress the proliferation, migration and invasiveness of human head and neck squamous cell carcinoma cells via p63­mediated tight junction molecules and p21­mediated growth arrest.

In human head and neck squamous cell carcinoma (HNSCC), the invasion and metastatic properties of cancer cells are promoted by junctional adhesion molecule­A (JAM­A) and claudin­1; these are epithelial tight junction molecules regulated by histone deacetylases (HDACs) and transcription factor p63. HDAC expression is reportedly upregulated in HNSCC, and HDAC inhibitors suppress cancer cell proliferation by initiating proliferative arrest or apoptosis. However, little is known of the anti­cancer mechanisms of HDAC inhibitors in HNSCC. Thus, in the present study, the HNSCC Detroit 562 cell line and primary cultured HNSCC cells were treated with HDAC inhibitors to investigate their effects in HNSCC. Higher expression of p63, HDAC1, JAM­A and claudin­1 was observed in HNSCC tissues compared with the adjacent dysplastic regions. In Detroit 562 cells, treatment with trichostatin A (TSA), an inhibitor of HDAC1 and 6, downregulated the expression of p63, JAM­A and claudin­1, and upregulated that of acetylated tubulin; conversely, p63 knockdown resulted in the downregulation of JAM­A and claudin­1. Collectively, inhibiting HDAC suppressed the migration and invasiveness of cancer cells. In addition, treatment with TSA suppressed cancer cell proliferation via G2/M arrest, as well as upregulating p21 and downregulating cyclin D1 expression. TSA also downregulated the expression of epidermal growth factor receptor (EGFR) and phospho­ERK1/2. p63 knockdown and treatment with an EGFR inhibitor induced G1 arrest and downregulated EGFR and phospho­ERK1/2 levels, respectively. HDAC inhibition also suppressed the migration and invasiveness of primary cultured HNSCC cells. Collectively, the results of the present study indicate that HDAC inhibitors suppress the proliferation, migration and invasiveness of HNSCC by downregulating the p63­mediated tight junction molecules JAM­A and claudin­1, and inducing p63 or p21­mediated growth arrest.

Increase in Epithelial Permeability and Cell Metabolism by High Mobility Group Box 1, Inflammatory Cytokines and TPEN in Caco-2 Cells as a Novel Model of Inflammatory Bowel Disease.

High mobility group box 1 protein (HMGB1) is involved in the pathogenesis of inflammatory bowel disease (IBD). Patients with IBD develop zinc deficiency. However, the detailed roles of HMGB1 and zinc deficiency in the intestinal epithelial barrier and cellular metabolism of IBD remain unknown. In the present study, Caco-2 cells in 2D culture and 2.5D Matrigel culture were pretreated with transforming growth factor-ß (TGF-ß) type 1 receptor kinase inhibitor EW-7197, epidermal growth factor receptor (EGFR) kinase inhibitor AG-1478 and a TNFα antibody before treatment with HMGB1 and inflammatory cytokines (TNFα and IFNγ). EW-7197, AG-1478 and the TNFα antibody prevented hyperpermeability induced by HMGB1 and inflammatory cytokines in 2.5D culture. HMGB1 affected cilia formation in 2.5D culture. EW-7197, AG-1478 and the TNFα antibody prevented the increase in cell metabolism induced by HMGB1 and inflammatory cytokines in 2D culture. Furthermore, ZnSO4 prevented the hyperpermeability induced by zinc chelator TPEN in 2.5D culture. ZnSO4 and TPEN induced cellular metabolism in 2D culture. The disruption of the epithelial barrier induced by HMGB1 and inflammatory cytokines contributed to TGF-ß/EGF signaling in Caco-2 cells. The TNFα antibody and ZnSO4 as well as EW-7197 and AG-1478 may have potential for use in therapy for IBD.

HMGB1 enhances epithelial permeability via p63/TGF-ß signaling in lung and terminal bronchial epithelial cells.

High mobility group box 1 (HMGB1) is involved in the induction of airway inflammation and injury in patients with chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis (IPF). HMGB1 increased by transforming growth factor-ß1 (TGF-ß1), impairs airway epithelial barrier function in the lung. In the present study, to investigate how HMGB1 affects the barrier of normal human lung epithelial (HLE) cells, monolayer cells (2D culture) and bronchial-like spheroid cells (2.5 D Matrigel culture), which have lumen formation, were pretreated with TGF-ß type I receptor kinase inhibitor EW-7197 before treatment with HMGB1. In 2D culture, treatment with HMGB1 decreased expression of angulin-1/LSR, TRIC and CLDN-1, -4, -7 and increased that of CLDN-2. Pretreatment with EW-7197 prevented the changes of all tight junction molecules induced by HMGB1. In 2.5D Matrigel culture, treatment with HMGB1 induced permeability of FITC-dextran (FD-4) into the lumen, whereas pretreatment with EW-7197 prevented the hyperpermeability of FD-4 into the lumen caused by HMGB1. In 2.5D Matrigel culture, knockdown of transcription factor p63 prevented the hyperpermeability induced by HMGB1 as well as pretreatment with EW-7197. In the 2D culture of HLE cells with HMGB1, knockdown of p63 increased the level of angulin-1/LSR and CLDN-4, while pretreatment with EW-7197 enhanced the increase of CLDN-4 induced by knockdown of p63. Immunohistochemical analysis of IPF, CLDN-2, HMGB1 and p63 revealed that their levels were higher in the regenerative epithelium of the terminal bronchial region than in normal epithelium. HMGB1 induces epithelial permeability of HLE cells via p63/TGF-ß signaling in normal lung and IPF.

Possibility of Targeting Claudin-2 in Therapy for Human Endometrioid Endometrial Carcinoma.

Claudin-2 (CLDN-2) is a leaky-type tight junction protein, and its overexpression increases tumorigenesis of some types of cancer cells. In the present study, to examine the possibility of targeting CLDN-2 in the therapy for endometrioid endometrial adenocarcinoma, we investigated the regulation and role of CLDN-2 in endometriosis and endometrioid endometrial adenocarcinoma. In endometrioid endometrial adenocarcinoma tissues, marked upregulation of CLDN-2 was observed together with malignancy, while in endometriosis tissues, a change in the localization of CLDN-2 was observed. In cells of the endometrial adenocarcinoma cell line Sawano, which highly express CLDN-2, downregulation of CLDN-2 induced by the siRNA upregulated the epithelial barrier and inhibited cell migration. Furthermore, the downregulation of CLDN-2 affected the cell cycle and inhibited cell proliferation. In Sawano cells cultured with high-glucose medium, CLDN-2 expression was downregulated at the mRNA and protein levels. The high-glucose medium upregulated the epithelial barrier, cell proliferation, and migration, and inhibited cell invasion. The histone deacetylase (HDAC) inhibitor tricostatin A (TSA), which has antitumor effects, downregulated CLDN-2 expression, cell proliferation, invasion, and migration, and upregulated the epithelial barrier. The mitochondrial respiration level, an indicator of cancer metabolism, was downregulated by CLDN-2 knockdown and upregulated by the high-glucose condition. Taken together, these results indicated that overexpression of CLDN-2 closely contributed to the malignancy of endometrioid endometrial adenocarcinoma. Downregulation of CLDN-2 via the changes of the glucose concentration and treatment with HDAC inhibitors may be important in the therapy for endometrial cancer.