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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.

Guanylate-binding protein-1 (GBP-1) is an interferon-inducible large GTPase involved in the epithelial barrier at tight junctions. To investigate the role of GBP-1 in the epithelial barrier, primary human salivary gland duct epithelial cells were treated with the the proinflammatory cytokines IFNγ, IL-1β, TNFα and the growth factor TGF-β. Treatment with IFNγ, IL-1β, or TNFα markedly enhanced GBP-1 and the epithelial barrier function, and induced not only CLDN-7 but also the tricellular tight junction molecule lipolysis-stimulated lipoprotein receptor (LSR). Knockdown of GBP-1 by its siRNA induced endocytosis of tight junction molecules, and prevented the increases of CLDN-7 and LSR with the upregulation of the epithelial barrier function induced by treatment with IFNγ or TNFα. Treatment with a PKCα inhibitor induced expression of GBP-1, CLDN-7 and LSR and enhanced the epithelial barrier function. In almost intact salivary gland ducts from patients with IgG4-related disease (IgG4-RD) indicated significant infiltration of IgG-positive plasma cells, expression of GBP-1, CLDN-7 and LSR was increased. These findings indicated that GBP-1 might play a crucial role in barrier function of normal human salivary gland duct epithelium and perform a preventive role in the duct epithelium of IgG4-RD disease., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Objective: Intranasal insulin administration has therapeutic potential for Alzheimer's disease and in intranasal administration across the nasal mucosa, the paracellular pathway regulated by tight junctions is important. The C-terminal fragment of Clostridium perfringens enterotoxin (C-CPE) binds the tight junction protein claudin and disrupts the tight junctional barrier without a cytotoxic effect. The C-CPE mutant called C-CPE 194 binds only to claudin-4, whereas the C-CPE 194 mutant called C-CPE m19 binds not only to claudin-4 but also to claudin-1., Methods: In the present study, to investigate the effects of C-CPE mutants on the tight junctional functions of human nasal epithelial cells (HNECs) and on the permeability of human recombinant insulin across the cells, HNECs were treated with C-CPE 194 and C-CPE m19., Results: C-CPE 194 and C-CPE m19 disrupted the barrier and fence functions without changes in expression of claudin-1, -4, -7, and occludin or cytotoxicity, whereas they transiently increased the activity of ERK1/2 phosphorylation. The disruption of the barrier function caused by C-CPE 194 and C-CPE m19 was prevented by pretreatment with the MAPKK inhibitor U0126. Furthermore, C-CPE 194 and C-CPE m19 significantly enhanced the permeability of human recombinant insulin across HNECs and the permeability was also inhibited by U0126., Conclusion: These findings suggest that C-CPE mutants 194 and m19 can regulate the permeability of insulin across HNECs via the MAPK pathway and may play a crucial role in therapy for the diseases such as Alzheimer's disease via the direct intranasal insulin administration.

Tight junctions (TJs) are necessary for salivary gland function and may serve as indicators of salivary gland epithelial dysfunction. IgG4-related disease (IgG4-RD) is a newly recognized fibro-inflammatory condition which disrupts the TJ associated epithelial barrier. The salivary glands are one of the most frequently involved organs in IgG4-RD, however, changes of the TJ associated epithelial barrier in salivary gland duct epithelium is poorly understood. Here, we investigated the regulation and function of TJs in human submandibular gland ductal epithelial cells (HSDECs) in normal and IgG4-RD. We examined submandibular gland (SMG) tissue from eight control individuals and 22 patients with IgG4-RD and established an HSDEC culture system. Immunohistochemistry, immunocytochemistry, western blotting, and measurement of transepithelial electrical resistance (TER) were performed. Claudin-4, claudin-7, occludin, and JAM-A were expressed at the apical side of the duct epithelium in submandibular gland (SMG) tissue and at the cell borders in HSDECs of normal and IgG4-RD. The expression and distribution of TJs in SMG tissue were not different in control individuals and patients with IgG4-RD in vivo and in vitro. Although interferon-gamma (IFNγ) generally disrupts the integrity and function of TJs, as manifested by decreased epithelial barrier function, IFNγ markedly increased the epithelial barrier function of HSDECs via upregulation of claudin-7 expression in HSDECs from patients with IgG4-RD. This is the first report showing an IFNγ-dependent increase in epithelial barrier function in the salivary gland duct epithelium. Our results provide insights into the functional significance of TJs in salivary gland duct epithelium in physiological and pathological conditions, including IgG4-RD.

The liver comprises hepatocytes and non-parenchymal cells such as bile duct epithelial cells. Claudin-4 and -7 are not expressed in hepatocytes under physiological conditions. It was reported that claudin-7 increased in human pulmonary fibroses. We therefore investigated claudin-4 and -7 expressions in human cirrhotic livers, in which hepatocyte proliferation is severely delayed. We examined liver tissues from 50 patients with liver tumors. The expression of claudin-4 and -7 in hepatocytes significantly increased with the grade of fibrosis, not with inflammatory activity, in the liver tissues of chronic hepatitis. The number of claudin-4- and -7-positive cells observed was greater than that of alpha-fetoprotein-positive hepatic progenitor cells. In primary cultures of mouse hepatocytes, the expression of claudin-4 and -7 was not induced by treatment with proinflammatory cytokines. In immunohistochemical analysis of liver tissues of 3,5-diethoxycarbonyl-1,4-dihydrocollidine-treated mice and primary cultures of mouse hepatocytes, the expression of claudin-4 and -7 increased with proliferation of progenitor cells. However, the claudin-4- and -7-positive cells were not always progenitor cells. Thus, claudin-4 and -7 were observed in hepatocytes of severely damaged mouse and human livers. These findings suggest that claudin-4- and -7-positive hepatocytes may exist during the process of differentiation from progenitor cells into mature hepatocytes.

The number of patients with uterine endometrial carcinoma, the cause of which involves sex hormones, has recently been growing rapidly because of increases in life expectancy and obesity. Tight junction proteins claudin-3 and -4 are receptors of Clostridium perfringens enterotoxin (CPE) and increase during endometrial carcinogenesis. In the present study of normal human endometrial epithelial (HEE) cells and the uterus cancer cell line Sawano, we investigate changes in the expression of tight junction proteins including claudin-3 and -4, the fence and barrier functions of the tight junction and the cytotoxic effects of CPE by sex hormones. In primary cultured HEE cells, treatment with progesterone (P4) but not estradiol (E2), induced claudin-1, -3, -4 and -7 and occludin, together with the downregulation of the barrier function but not the fence function. In Sawano cells, claudin-3 and -4 were upregulated by E2 but not by P4, together with a disruption of both the barrier and fence function. In primary cultured HEE cells, claudin-3 and -4 were localized at the apicalmost regions (tight junction areas) and no cytotoxicity of CPE was observed. In Sawano cells, claudin-3 and -4 were found not only in the apicalmost regions but also at the basolateral membrane and the cytotoxicity of CPE was enhanced by E2. Thus, tight junctions are physiological regulated by sex hormones in normal HEE cells during the menstrual cycle suggesting that safer and more effective therapeutic methods targeting claudins in uterine cancer can be developed.

Ovarian adenocarcinomas, like human ovarian surface epithelial cells, form functional tight junctions. Tight junction molecules claudin-3 and claudin-4, which are the receptors of Clostridium perfringens enterotoxin (CPE), are abnormally upregulated in epithelial ovarian cancers of all subtypes including, mucinous cystadenocarcinoma and serous cystadenocarcinoma. Clostridium perfringens enterotoxin may be a novel tumor-targeted therapy for ovarian cancers. In epithelial ovarian cancers, overexpression of epidermal growth factor receptor has been observed and the exogenous ligand EGF induces epithelial-mesenchymal transition in ovarian surface epithelium. Epidermal growth factor (EGF) signaling modulates expression of claudins with changes of fence and barrier functions in various cell types. However, the regulation of tight junctions by EGF in ovarian cancers remains unclear. In the present study, to investigate the mechanisms of the regulation of tight junctions in ovarian cancers, ovarian cancer cell lines mucinous cystadenocarcinoma (MCAS) and serous cystadenocarcinoma (HUOA) were treated with EGF. Epidermal growth factor downregulated claudin-3 in MCAS and claudin-4 in HUOA by inducing degradation of the proteins with changes in structures and functions of tight junctions via the MEK/ERK or PI3K/Akt signaling pathway. In addition, in HUOA but not MCAS, EGF downregulated the cytotoxic effect of CPE via claudin-4. Thus, there were different mechanisms for regulation of claudins by EGF between subtypes of epithelial ovarian cancer cells in vitro. These results indicate that EGF may affect claudins and tight junctional functions in ovarian cancer cells during cancer progression.

To investigate the regulation of tight junction molecules in normal human pancreatic duct epithelial (HPDE) cells and pancreatic cancer cells, we introduced the human telomerase reverse transcriptase (hTERT) gene into HPDE cells in primary culture and compared them to pancreatic cancer cell lines. The hTERT-transfected HPDE cells were positive for PDE markers and expressed claudin-1, claudin-4, claudin-7, and claudin-18, occludin, tricellulin, marvelD3, JAM-A, zonula occludens (ZO)-1, and ZO-2. The tight junction molecules, including claudin-4 and claudin-18 of normal HPDE cells, were in part regulated via a protein kinase C signal pathway by transcriptional control. In addition, claudin-18 in normal HPDE cells and pancreatic cancer cells was markedly induced by a PKC activator, and claudin-18 in pancreatic cancer cells was also modified by DNA methylation. In the marvel family of normal HPDE cells and pancreatic cancer cells, tricellulin was upregulated via a c-Jun N-terminal kinase pathway, and marvelD3 was downregulated during Snail-induced epithelial-mesenchymal transition., (© 2012 New York Academy of Sciences.)

Background: Clostridium perfringens enterotoxin (CPE) triggers lysis of epithelial cells through binding to tight-junction proteins claudin-3 (Cldn3) and Cldn4, which are over-expressed in prostate cancer. We investigated the potential of Cldn-targeted therapy using CPE., Methods: We investigated the expression levels and subcellular localization of Cldn3 and Cldn4 in primary human prostate cancer tissues, human prostate cancer cell lines (22Rv1, DU145, and PC3) and normal human prostate epithelial cells (PrECs). Cytotoxic effects of CPE on these cells were examined by colorimetric assay. We studied whether knockdown of Cldn3 and/or Cldn4 expression using RNA interference influenced CPE-mediated cytotoxicity. The therapeutic effect of CPE was evaluated in PC3 xenografts in athymic mice., Results: Cldn4 and Cldn3 were expressed in primary human prostate cancer tissues, 22Rv1, DU145, and PC3. Cldn4 protein was expressed in PrEC. Cldn4 was distributed along whole cell membranes of the cancer cell lines, whereas it was localized at tight junctions in PrEC. CPE-mediated cytotoxicity was greatly detected in PC3, but was hardly detectable in PrEC. Reduced expression of Cldn4, but not Cldn3, led to remarkable decreases of cytotoxicity in both PC3 and 22Rv1. The injection of CPE around PC3 xenografts significantly suppressed tumor growth., Conclusion: CPE-mediated cytotoxicity was observed in human prostate cancer cell lines, but barely detected in normal human PrECs. The cytotoxic effect depended not only on the expression level of Cldn4 protein but also on its subcellular localization. These results suggest that Cldn4-targeted therapy using CPE may be a new treatment for prostate cancer., (Copyright © 2011 Wiley Periodicals, Inc.)

Protein kinase C (PKC) is overexpressed in cancer, including pancreatic cancer, compared with normal tissue. Moreover, PKCα is considered one of the biomarkers for the diagnosis of cancers. In several human cancers, the claudin tight junction molecules are abnormally regulated and are thus promising molecular targets for diagnosis and therapy with Clostridium perfringens enterotoxin (CPE). In order to investigate the changes of tight junction functions of claudins via PKCα activation in pancreatic cancer cells, the well-differentiated human pancreatic cancer cell line HPAC, with its highly expressed tight junction molecules and well-developed barrier function, was treated with the PKC activator 12-O-tetradecanoylphorbol 13-acetate (TPA). Treatment with TPA modified the activity of phosphoPKCα and caused an increase of the Snail family members Snail, Slug and Smad-interacting protein 1 and a decrease of E-cadherin. In HPAC cells treated with TPA, downregulation of claudin-1 and mislocalization of claudin-4 and occludin around the nuclei were observed, together with a decrease in the numbers of tight junction strands and an increase in phosphorylation of claudin-4. The barrier function and the cytotoxicity of CPE were significantly decreased on TPA treatment. All such changes after TPA treatment were prevented by inhibitors of panPKC and PKCα. These findings suggest that, in human pancreatic cancer cells, PKCα activation downregulates tight junction functions as a barrier and as a receptor of CPE via the modification of claudin-1 and -4 during epithelial to mesenchymal transition-like changes. PKCα inhibitors might represent potential therapeutic agents against human pancreatic cancer cells by use of CPE cytotoxicity via claudin-4.

The tight junction protein claudin-4 is frequently overexpressed in pancreatic cancer, and is also a receptor for Clostridium perfringens enterotoxin (CPE). The cytotoxic effects of CPE are thought to be useful as a novel therapeutic tool for pancreatic cancer. However, the responses to CPE via claudin-4 remain unknown in normal human pancreatic duct epithelial (HPDE) cells. We introduced the human telomerase reverse transcriptase (hTERT) gene into HPDE cells in primary culture as a model of normal HPDE cells in vitro. hTERT-HPDE cells treated with or without 10% FBS and pancreatic cancer cell lines PANC-1, BXPC3, HPAF-II and HPAC were treated with CPE. In Western blotting, the expression of claudin-4 protein in hTERT-HPDE cells treated with 10% FBS was as high as it was in all of the pancreatic cancer cell lines. In hTERT-HPDE cells with or without 10% FBS, cytotoxicity was not observed at any concentration of CPE, whereas in all pancreatic cancer cell lines, CPE had a dose-dependent cytotoxic effect. In hTERT-HPDE cells with 10% FBS, claudin-4 was localized in the apical-most regions, where there are tight junction areas, in which in all pancreatic cancer cell lines claudin-4 was found not only in the apical-most regions but also at basolateral membranes. In hTERT-HPDE cells with 10% FBS after treatment with CPE, downregulation of barrier function and claudin-4 expression at the membranes was observed. In HPAC cells, the sensitivity to CPE was significantly decreased by knockdown of claudin-4 expression using siRNA compared to the control. These findings suggest that, in normal HPDE cells, the lack of toxicity of CPE was probably due to the localization of claudin-4, which is different from that of pancreatic cancer cells. hTERT-HPDE cells in this culture system may be a useful model of normal HPDE cells not only for physiological regulation of claudin-4 expression but also for developing safer and more effective therapeutic methods targeting claudin-4 in pancreatic cancer.

Conclusions: Altered expression of claudin-1, claudin-7, and tricellulin in early tonsillar squamous cell carcinoma (SCC) independent of human papilloma virus (HPV) infection may lead to tumor progression., Objectives: Integral tight junction proteins, the claudins and tricellulin, play a crucial role in all tissues. HPV is significantly associated with tonsillar SCC. We sought to determine the expression of claudin-1, claudin-7, and tricellulin in HPV-infected and HPV-free tonsillar SCC., Methods: Twenty-eight tonsillar SCCs were studied by immunohistochemical analysis and real-time reverse transcription polymerase chain reaction with in situ hybridization analysis., Results: Compared with normal tissues, claudin-1 was strongly expressed, whereas claudin-7 and tricellulin were weakly expressed or were absent in primary SCC and metastatic lymph nodes. Claudin-7 and tricellulin were markedly reduced at all stages of tumor development. In situ hybridization analysis showed no correlation between HPV infection and altered expression of the tight junction proteins.

Tight junctions of hepatocytes play crucial roles in the barrier to keep bile in bile canaliculi away from the blood circulation, which we call the blood-biliary barrier. Tight junction proteins of hepatocytes are regulated by various cytokines and growth factors via distinct signal transduction pathways. To investigate changes in expression and function of tight junction proteins including claudins via signal transduction pathways in hepatocytes during EMT induced by TGF-β, we examined effects of TGF-β on expression and localization of the integral tight junction proteins, claudin-1, -2, and occludin, as well as the fence function by using primary cultures of adult rat hepatocytes.

Epithelial-derived thymic stromal lymphopoietin (TSLP) triggers dendritic cell (DC)-mediated Th2-type inflammatory responses and is a master switch for allergic inflammatory diseases. In the present study, the expression and induction of TSLP and the effects of TSLP on the tight-junctional barrier of human nasal epithelial cells (HNECs) have been investigated in order to elucidate the role of TSLP in allergic rhinitis. We have found high expression of TSLP in the epithelium from patients with allergic rhinitis with recruitment and infiltration of DCs. In vitro, TSLP is significantly produced in HNECs after treatment with a toll-like receptor 2 (TLR2) ligand, Pam(3)Cys-Ser-(Lys)(4), and a mixture of interleukin-1beta and tumor necrosis factor-alpha. Treatment with TSLP rapidly enhances the barrier function of cultured HNECs, together with an increase of tight-junction proteins claudin-1, -4, -7, and occludin. The nasal-epithelial-derived TSLP thus not only activates DCs but also preserves the epithelial barrier via the upregulation of tight-junction proteins, thereby regulating antigen sensitization during the early stage of allergic rhinitis.

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 2β 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

P63 is a regulator of cell-cell junction complexes in the epidermis. Claudin-4 is regulated via various factors in normal epithelial cells and diseases. We found that claudin-4 was directly regulated via p63 (TAp63 and ΔNp63) in human keratinocytes and nasal epithelial cells. In the epidermis of atopic dermatitis (AD), which contains ΔNp63-deficient keratinocytes, high expression of claudin-4 was observed. In primary keratinocytes, downregulation of ΔNp63 by treatment with short interfering RNA (siRNA)-p63 induced claudin-4 expression. In nasal epithelial cells in the context of rhinitis or nasal polyps, upregulation of TAp63 and downregulation of claudin-4 were observed. In primary nasal epithelial cells transfected with the human telomerase reverse transcriptase gene, knockdown of p63 by siRNAs induced claudin-4 expression. Taken together, these findings indicate that p63 is a negative regulator of claudin-4 expression. Understanding the regulation of claudin-4 via p63 in human epithelial cells may be important for developing therapies for allergies and drug delivery systems. [ABSTRACT FROM AUTHOR]

DDC (3,5-diethoxycarbonyl-1,4-dihydrocollidine)-fed mice are widely used as a model for cholestatic liver disease. We examined the expression of tight junction protein claudin subspecies by immunofluorescent histochemistry in small intestine and kidney tissues of mice fed a DDC diet for 12 weeks. In the small intestine, decreases in claudin-3, claudin-7 and claudin-15 were observed in villous epithelial cells corresponding to the severity of histological changes while leaving the abundance of these claudin subspecies unchanged in crypt cells. Nevertheless, the proliferative activity of intestinal crypt cells measured by immunohistochemistry for Ki- 67 decreased in the mice fed the DDC diet compared with that of control mice. These results suggest the possibility that DDC feeding affects the barrier function of villous epithelial cells and thus inhibits the proliferative activity of crypt epithelial cells. On the other hand, in the kidney, remarkable changes were found in the subcellular localization of claudin subspecies in a segment-specific manner, although histological changes of renal epithelial cells were quite minimal. These results indicate that immunohistochemistry for claudin subspecies can serve as a useful tool for detecting minute functional alterations of intestinal and renal epithelial cells. [ABSTRACT FROM AUTHOR]

Protein kinase Cα (PKCα) is highly expressed in pancreatic cancer. However, the effects of PKCα on Snail and claudin-1, which play crucial roles in epithelial cell polarity during epithelial–mesenchymal transition (EMT), remain unclear. In this study, we investigated the mechanisms of regulation of Snail and claudin-1 via a PKCα signal pathway during EMT in pancreatic cancer cells and in normal human pancreatic duct epithelial cells (HPDEs). By immunostaining, overexpression of PKCα and downregulation of claudin-1 were observed in poorly differentiated human pancreatic cancer tissues and the pancreatic cancer cell line PANC-1. Treatment with the PKCα inhibitor Gö6976 transcriptionally decreased Snail and increased claudin-1 in PANC-1 cells. The PKCα inhibitor prevented upregulation of Snail and downregulation of claudin-1 during EMT induced by transforming growth factor-β1 (TGF-β1) treatment and under hypoxia in PANC-1 cells. The effects of the PKCα inhibitor were in part regulated via an extracellular signal-regulated kinase (ERK) signaling pathway. The PKCα inhibitor also prevented downregulation of the barrier function and fence function during EMT in well-differentiated pancreatic cancer cell line HPAC. In normal HPDEs, the PKCα inhibitor transcriptionally induced not only claudin-1 but also claudin-4, -7 and occludin without a change of Snail. Treatment with the PKCα inhibitor in normal HPDEs prevented downregulation of claudin-1 and occludin by TGF-β1 treatment and enhanced upregulation of claudin-1, -4, -7 and occludin under hypoxia. These findings suggest that PKCα regulates claudin-1 via Snail- and mitogen-activated protein kinase/ERK-dependent pathways during EMT in pancreatic cancer. Thus, PKCα inhibitors may be potential therapeutic agents against the malignancy of human pancreatic cancer cells. [ABSTRACT FROM PUBLISHER]

Simple Summary: Abnormality of tight junction proteins closely contributes to epithelial–mesenchymal transition (EMT) and the malignancy of various cancers. Angulin-1/lipolysis-stimulated lipoprotein receptor (LSR) is a novel molecular constituent of tricellular contacts that has a barrier function. Loss of angulin-1/LSR correlates with the malignancy in various cancers, including endometrioid-endometrial carcinoma (EEC). Moreover, loss of angulin-1/LSR upregulates claudin-1, and loss of apoptosis-stimulating p53 protein 2 (ASPP2) at tricellular contacts downregulates angulin-1/LSR in human EEC cell line Sawano. Angulin-1/LSR and ASPP2 concentrate at both midbody and centrosome during cytokinesis in Sawano. 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 the loss of angulin-1/LSR promotes the progression of endometriosis and EEC and discusses the possibility of therapeutic targeting for angulin-1/LSR via multiple signaling pathways and its related proteins. 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. [ABSTRACT FROM AUTHOR]

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-β1 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-β 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-β signaling in HNECs. [ABSTRACT FROM AUTHOR]

In the epidermis, tight junction (TJ) structure is specifically located in the stratum granulosum, where the expression of ΔNp63, a p53 family transcription factor, is attenuated. Since the relationship between ΔNp63 and barrier function has not been fully uncovered, we assessed expression profiles of TJ proteins in skin tissues and cultured keratinocytes. The results showed that expression of ΔNp63 and that of claudin-4 were inversely correlated in healthy human epidermis. In vitro studies using HaCaT keratinocytes revealed functional relevance of ΔNp63 and claudin-4. Curiously, Toll-like receptor (TLR)-3 ligand, which is known to be liberated from damaged cells, suppressed ΔNp63 expression and concomitantly upregulated claudin-4 expression in primary keratinocytes. More interestingly, a broad expression pattern of claudin-4 was found in the epidermis of atopic dermatitis (AD), a barrier defect disorder, which contains ΔNp63-lacking keratinocytes as we reported previously. Therefore, upregulation of claudin-4 expression regulated by ΔNp63 might be associated with complementary or repair responses of damaged keratinocytes with AD. [ABSTRACT FROM AUTHOR]

Cyclic AMP (cAMP) promotes functions of tight junctions in endothelial cells, although its target remains unknown. We showed here that cAMP increased gene expression of claudin-5 and decreased that of claudin-1 in porcine blood–brain-barrier endothelial cells via protein kinase A (PKA)-independent and -dependent pathways, respectively. cAMP also enhanced immunoreactivity of claudin-5 along cell borders and in the cytoplasm, reorganized actin filaments, and altered signals of claudin-5, occludin, ZO-1, and ZO-2 along cell boundaries from zipperlike to linear patterns. In contrast, claudin-1 was detected only in the cytoplasm in a dotlike pattern, and its immunolabeling was reduced by cAMP. Interestingly, 31- and 62-kDa claudin-5 immunoprecipitates in the NP-40-soluble and -insoluble fractions, respectively, were highly phosphorylated on threonine residue(s) upon cAMP treatment. All these changes induced by cAMP, except for claudin-5 expression and its signals in the cytoplasm, were reversed by an inhibitor of PKA, H-89. We also demonstrated that cAMP elevated the barrier function of tight junctions in porcine blood–brain-barrier endothelial cells in PKA-dependent and -independent manners. These findings indicate that both PKA-induced phosphorylation of claudin-5 immunoprecipitates and cAMP-dependent but PKA-independent induction of claudin-5 expression could be involved in promotion of tight-junction function in endothelial cells. [Copyright &y& Elsevier]

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)-4α, a nuclear receptor. We herein show that induction of HNF-4α 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-4α. In contrast, expression levels of ZO-1, ZO-2, E-cadherin, and β-catenin were not altered by HNF-4α. We also demonstrate, by analysis of diffusion of labeled sphingomyelin, that the fence function of tight junctions is achieved by induction of HNF-4α. These findings indicate that HNF-4α triggers de novo formation of functional tight junctions and establishment of epithelial cell polarity. [Copyright &y& Elsevier]

A non-histone chromatin-associated protein, high mobility group box 1 (HMGB1), which impairs the airway epithelial barrier, is involved in the induction of airway inflammation in patients with allergy, asthma, chronic obstructive pulmonary disease (COPD), and idiopathic pulmonary fibrosis (IPF). Tricellular tight junctions (tTJs) form at the convergence of bicellular tight junctions (bTJs). Angulin-1/lipolysis-stimulated lipoprotein receptor (LSR) is a novel molecule present at tricellular contacts and contributes to the epithelial barrier and cellular metabolism. Adenosine monophosphate-activated protein kinase (AMPK) is a central metabolic regulator and has a reciprocal association with TJs. In the present study, to examine how HMGB1 contributes to airway epithelial barrier disruption and the cellular metabolism indicated as mitochondrial respiration, bronchial epithelial Calu-3 cells were transfected with siRNAs of angulin-1/LSR or treated with HMGB1 and the relationship between HMGB1 and angulin-1/LSR was investigated. Knockdown of angulin-1/LSR upregulated the expression of the tight junction molecule claudin-2, AMPK activity, and mitochondrial respiration, and downregulated the epithelial barrier. Treatment with HMGB1 downregulated angulin-1/LSR expression and the epithelial barrier, and upregulated claudin-2 expression, AMPK activity and mitochondrial respiration. Treatment with EW-7197, a transforming growth factor-β (TGF-β) type I receptor kinase inhibitor, prevented all the effects of HMGB1 in Calu-3 cells. HMGB1-downregulated angulin-1/LSR induced epithelial barrier disruption via claudin-2 and cellular metabolism via AMPK in airway epithelial Calu-3 cells. The effects of HMGB1 contribute to TGF-β signaling and EW-7197 shows potential for use in therapy for HMGB1-induced airway inflammation. •HMGB1-impaired airway epithelial barrier is involved in airway inflammation. •Angulin-1/LSR contributes to the airway epithelial barrier and cellular metabolism. •AMPK has a reciprocal association with cellular metabolism and tight junctions. • Downregulation of angulin-1/LSR induces claudin-2 and AMPK activity. •HMGB1-downregulated LSR induces barrier disruption and cellular metabolism. [ABSTRACT FROM AUTHOR]

The epithelial-mesenchymal transition (EMT) is an essential step in cancer progression. Epithelial cells possess several types of cell-cell junctions, and tight junctions are known to play important roles in maintaining the epithelial program. EMT is characterized by a loss of epithelial markers, including E-cadherin and tight junction proteins. Somewhat surprisingly, the evidence is accumulating that upregulated expression of tight junction proteins plays an important role in the EMT of cancer cells. Tight junctions have distinct tissue-specific and cancer-specific regulatory mechanisms, enabling them to play different roles in EMT. Tight junctions and related signaling pathways are attractive targets for cancer treatments; signal transduction inhibitors and monoclonal antibodies for tight junction proteins may be used to suppress EMT, invasion, and metastasis. Here we review the role of bicellular and tricellular tight junction proteins during EMT. Further investigation of regulatory mechanisms of tight junctions during EMT in cancer cells will inform the development of biomarkers for predicting prognosis as well as novel therapies. • Several tight junction molecules promote or suppress EMT of cancer cells. • Tight junction proteins regulate intracellular signaling depending on their localization and cancer type. • Tight junction proteins on the basolateral membrane and in the nucleus have different roles and may promote EMT of cancer. • Tight junctions and related signaling are promising targets of diagnostic and therapeutic tools against cancer. • Specific antibodies to the extracellular domain of claudins may become novel anti-cancer drugs. [ABSTRACT FROM AUTHOR]