Takayuki Kohno

Pseudomonas aeruginosa elastase causes transient disruption of tight junctions and downregulation of PAR-2 in human nasal epithelial cells.

BackgroundPseudomonas aeruginosa causes chronic respiratory disease, and the elastase enzyme that it produces increases the permeability of airway epithelial cells owing to the disruption of tight junctions. P. aeruginosa is also implicated in prolonged chronic rhinosinusitis. However, the effects of P. aeruginosa elastase (PE) against the barrier formed by human nasal epithelial cells (HNECs) remain unknown.MethodsTo investigate the mechanisms involved in the disruption of tight junctions by PE in HNECs, primary cultures of HNECs transfected with human telomerase reverse transcriptase (hTERT-HNECs) were used. The hTERT-HNECs were pretreated with inhibitors of various signal transduction pathways, PKC, MAPK, p38MAPK, PI3K, JNK, NF-κB, EGF receptor, proteasome, COX1 and COX2 before treatment with PE. Some cells were pretreated with siRNA and agonist of protease activated receptor-2 (PAR-2) before treatment with PE. Expression and structures of tight junctions were determined by Western blotting, real-time PCR, immunostaining and freeze-fracture. Transepithelial electrical resistance (TER) was examined as the epithelial barrier function.ResultsPE treatment transiently disrupted the epithelial barrier and downregulated the transmembrane proteins claudin-1 and -4, occludin, and tricellulin, but not the scaffold PDZ-expression proteins ZO-1 and -2 and adherens junction proteins E-cadherin and β-catenin. The transient downregulation of tight junction proteins was controlled via distinct signal transduction pathways such as the PKC, MAPK, PI3K, p38 MAPK, JNK, COX-1 and -2, and NF-κB pathways. Furthermore, treatment with PE transiently decreased PAR-2 expression, which also regulated the expression of the tight junction proteins. Treatment with a PAR-2 agonist prevented the downregulation of the tight junction proteins after PE treatment in HNECs.ConclusionsPE transiently disrupts tight junctions in HNECs and downregulates PAR-2. The transient disruption of tight junctions by PE might occur repeatedly during chronic rhinosinusitis.

Poly(I:C) induced microRNA-146a regulates epithelial barrier and secretion of proinflammatory cytokines in human nasal epithelial cells

Human nasal epithelial cells (HNECs) are important in the tight junctional barrier and innate immune defense protecting against pathogens invading via Toll-like receptors (TLRs). MicroRNAs (miRNAs) regulate expression of tight junctions as direct or indirect targeting genes and maintain the barrier function. However, the roles of miRNAs in the epithelial barrier of HNECs via TLRs remain unknown. In the present study, to investigate the effects of miRNAs on the epithelial barrier of HNECs via TLRs, primary cultured HNECs transfected with human telomerase reverse transcriptase (hTERT-HNECs), were treated with the TLR3 ligand poly(I:C) and miRNA array analysis was performed. In the miRNA array of the cells treated with poly(I:C), upregulation of miR-187, -146a, -574, -4274, -4433, -4455 and -4750, and downregulation of miR-4785 by more than twofold compared to the control were observed. When control HNECs were treated with mimics and inhibitors of these miRNAs, an miR-146a mimic induced expression of tight junction proteins claudin-1, occludin and JAM-A together with an increase of the epithelial barrier function. The poly(I:C)-induced miR-146a was regulated via the distinct TLR3-mediated signal pathways PI3K, JNK and NF-κB. Furthermore, the miR-146a mimic prevented downregulation of claudin-1 and JAM-A and the secretion of proinflammatory cytokines IL-8 and TNF-α induced by poly(I:C) by targeting TRAF6. These findings indicate that, in HNECs, miRNA-146a plays crucial roles in maintenance of the tight junction barrier and innate immune defense protecting against invading pathogens.

Expression of tricellulin in epithelial cells and non-epithelial cells.

Tricellulin is the first molecular component of tricellular tight junctions at tricellular contacts where three epithelial cells meet, and it is required for the their formation and maintenance of the epithelial barrier. Tricellulin binds other tight junction proteins, and its expression and distribution are affected by the bicellular tight junction protein occludin and lipolysis-stimulated lipoprotein receptor (LSR) which is expressed at tricellular contacts. Tricellulin is also detected in endothelial cells, neurons, microglia and astrocytes. Here, we focused tricellulin expression in various types of epithelial cells, nasal epithelial cells, pancreatic duct epithelial cells cells and hepatocytes, and non-epithelial cells, dendritic cells and Schwann cells, compared to expression of the bicellular tight junction protein occludin and LSR, and discuss the regulation and the role of tricellulin in cellular specificity.

Claudin-binder C-CPE mutants enhance permeability of insulin across human nasal epithelial cells

Abstract Objective: Intranasal insulin administration has therapeutic potential for Alzheimers 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 Alzheimers disease via the direct intranasal insulin administration.

The roles of tricellular tight junction protein lipolysis-stimulated lipoprotein receptor in malignancy of human endometrial cancer cells

Lipolysis-stimulated lipoprotein receptor (LSR) has been identified as a novel molecular constituent of tricellular contacts that have a barrier function for the cellular sheet. LSR recruits tricellulin (TRIC), which is the first molecular component of tricellular tight junctions. Knockdown of LSR increases cell motility and invasion of certain cancer cells. However, the behavior and the roles of LSR in endometrial cancer remain unknown. In the present study, we investigated the behavior and roles of LSR in normal and endometrial cancer cells in vivo and in vitro. In endometriosis and endometrial cancer, LSR was observed not only in the subapical region but also throughout the lateral region as well as in normal endometrial epithelial cells in the secretory phase, and LSR in the cancer was reduced in correlation with the malignancy. Knockdown of LSR by the siRNA in cells of the endometrial cancer cell line Sawano, induced cell migration, invasion and proliferation, while TRIC relocalized from the tricellular region to the bicellular region at the membrane. In Sawano cells and normal HEEs, a decrease of LSR induced by leptin and an increase of LSR induced by adiponectin and the drugs for type 2 diabetes metformin and berberine were observed via distinct signaling pathways including JAK2/STAT. In Sawano cells, metformin and berberine prevented cell migration and invasion induced by downregulation of LSR by the siRNA and leptin treatment. The dissection of the mechanism in the downregulation of endometrial LSR during obesity is important in developing new diagnostic and therapy for endometrial cancer.

c-Jun N-terminal kinase inhibitor SP600125 enhances barrier function and elongation of human pancreatic cancer cell line HPAC in a Ca-switch model.

Abstractc-Jun N-terminal kinase (JNK), known as a stress-activated protein kinase, regulates normal epithelial biological processes, including assembly of adherens and tight junctions, and it is involved in the development of several cancers. The JNK inhibitor SP600125 enhances epithelial barrier function through modulation of tight junction molecules in normal human pancreatic epithelial cells. Furthermore, this JNK inhibitor suppresses the growth of human pancreatic cancer cells. However, the effects of SP600125 on the epithelial barrier in human pancreatic cancer cells remain unknown. In the present study, the JNK inhibitor SP600125 markedly enhanced the barrier function and cell elongation of well-differentiated human pancreatic cancer cell line HPAC in a Ca-switch model. The epithelial barrier function induced by SP600125 was regulated by phosphorylated β-catenin without changes in the tight junction molecules. The cell elongation induced by SP600125 was closely related to the expression of the F-actin-binding protein DrebrinE. These findings suggest that JNK is involved in the regulation of the epithelial barrier function and cell shape during remodeling of pancreatic cancer cells. The JNK inhibitor SP600125 may have potential as a therapeutic drug for pancreatic cancer via induction of differentiation.

FLT3/ ITD regulates leukaemia cell adhesion through α4β1 integrin and Pyk2 signalling

Internal tandem duplication of FMS‐like receptor tyrosine kinase 3 (FLT3/ITD) within its juxtamembrane domain is a frequent mutation in adult acute myeloid leukaemia (AML). This mutation causes constitutive activation of FLT3 and is associated with poor prognosis. The high relapse rate of FLT3/ITD‐positive AML might be partly because of insufficient eradication of slow‐cycling leukaemic stem cells in the bone marrow microenvironment. β1 integrin mediates haematopoietic stem and progenitor cell homing along with their retention in the bone marrow and also inhibits haematopoietic proliferation and differentiation. Here, we demonstrate that inhibition of FLT3/ITD kinase activity by a FLT3 selective inhibitor named FI‐700 decreases affinity of α4β1 integrin to soluble VCAM‐1. α4β1 integrin deactivation by FI‐700 is independent of Rap1, which is the critical regulator of integrin inside‐out signalling. In addition, selective inhibition of FLT3/ITD induces Pyk2 dephosphorylation together with the inhibition of phosphatidylinositol‐3‐kinase (PI3K)/Akt pathway. Both wild‐type and ITD‐FLT3 proteins co‐immunoprecipitated with β1 integrin and Pyk2 indicating the signal crosstalk between FLT3, β1 integrin and Pyk2. These results collectively indicated that the inhibition of FLT3 kinase might contribute not only to the induction of apoptosis, but also to the leukaemia cell detachment from the bone marrow microenvironment in the treatment of AML.

Dysregulation of junctional adhesion molecule-A via p63/GATA-3 in head and neck squamous cell carcinoma

Junctional adhesion molecule-A (JAM-A), which belongs to the IgG superfamily, is a tight junction molecule associated with epithelial and endothelial barrier function. Overexpression of JAM-A is also closely associated with invasion and metastasis of cancers such as breast cancer, lung cancer and pancreatic cancer. However, little is known about the mechanism in overexpression of JAM-A in head and neck squamous cell carcinoma (HNSCC). In the present study, we found high expression of JAM-A at the protein and mRNA levels in HNSCC tissues, including those of the oropharynx, larynx, and hypopharynx, together with high protein expression of β-catenin, p63, ΔNp63 and GATA-3. Furthermore, in ELISA, a significant increase of soluble JAM-A in the sera of HNSCC patients was observed compared to healthy subjects. Knockdown of JAM-A by siRNA inhibited cell proliferation, invasion and migration in the HNSCC cell line Detroit562 in vitro. JAM-A expression in Detroit562 was increased via a distinct signal transduction pathway including NF-κB. Expression of JAM-A, β-catenin, p63 and ΔNp63 in Detroit562 was decreased under hypoxia. Knockdown of p63, ΔNp63 or GATA-3 by siRNAs reduced JAM-A expression in Detroit562. In primary cultured HNSCC cells in which CK7, p63, ΔNp63 and GATA-3 were detected, JAM-A expression was decreased by knockdown of p63 or ΔNp63. These results indicate that JAM-A is a biomarker of malignancy in HNSCC and that plasma soluble JAM-A may contribute to serum-based diagnosis of HNSCC. The mechanism of dysregulation of JAM-A via p63/GATA-3 is important in possible molecular targeted therapy for HNSCC.

Claudin‐4 binder C‐CPE 194 enhances effects of anticancer agents on pancreatic cancer cell lines via a MAPK pathway

The C‐terminal fragment of Clostridium perfringens enterotoxin (C‐CPE) modulates the tight junction protein claudin and disrupts the tight junctional barrier. It also can enhance the effectiveness of anticancer agents. However, the detailed mechanisms of the effects of C‐CPE remain unclear in both normal and cancerous cells. The C‐CPE mutant called C‐CPE 194 binds only to claudin‐4, but the C‐CPE 194 mutant called C‐CPE m19 binds not only to claudin‐4 but also to claudin‐1. In the present study, to investigate the mechanisms of the effects of C‐CPE on claudin expression, the tight junctional functions and the cytotoxicity of anticancer agents, human pancreatic cancer cells, and normal human pancreatic duct epithelial cells (HPDEs) were treated with C‐CPE 194 and C‐CPE m19. In well‐differentiated cells of the pancreatic cancer cell line HPAC, C‐CPE 194 and C‐CPE m19 disrupted both the barrier and fence functions without changes in expression of claudin‐1 and ‐4, together with an increase of MAPK phosphorylation. C‐CPE 194, but not C‐CPE m19, enhanced the cytotoxicity of the anticancer agents gemcitabine and S‐1. In poorly differentiated pancreatic cancer cell line PANC‐1, C‐CPE 194, but not C‐CPE m19, decreased claudin‐4 expression and enhanced MAPK activity and the cytotoxicity of the anticancer agents. In normal HPDEs, C‐CPE 194 and C‐CPE m19 decreased claudin‐4 expression and enhanced the MAPK activity, whereas they did not affect the cytotoxicity of the anticancer agents. Our findings suggest that the claudin‐4 binder C‐CPE 194 enhances effects of anticancer agents on pancreatic cancer cell lines via a MAPK pathway.

Regulation of claudin‐4 via p63 in human epithelial cells

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.