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.

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.

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.

Mechanism of fibrogenesis in submandibular glands in patients with IgG4-RD.

The aim of this study was to investigate the mechanisms driving fibrosis in the submandibular glands (SMG) of patients with IgG4-related disease (IgG4-RD). Immunohistochemistry showed that many fibroblast-like cells expressing IL-6, IL-18, TSLP, IL-33, and MMP1 were present in SMG from the affected patients. SMG fibroblasts were derived from patients with or without IgG4-RD and were cultured in vitro. Expression of IL-6, IL-18, TSLP, IL-33 and MMP1, the secretion of IL-6 and G2/M phase were upregulated in the fibroblasts from the affected patients. By treatment with inflammatory cytokines IL-1ß, TNFα or TGF-ß after treatment with or without the NF-κB inhibitor curcumin, curucumin blocked the production and secretion of IL-6 upregulated by IL-1ß, TNFα, or TNFα/TGF-ß in all fibroblasts. Wnt1-inducible signaling protein 1 (WISP1), which can enhance fibroblasts proliferation, was also more abundantly expressed in affected fibroblasts, while treatment with IL-6 induced WISP1, treatment with WISP1 increased the G2/M phase, and curucumin inhibited WISP1 induced by TNFα/TGF-ß in unaffected fibroblasts. IL-33 in affected fibroblasts was induced by IL-1ß, TNFα, or TNFα/TGF-ß, while the effect of IL-1ß or TNFα/TGF-ß was blocked by curcumin. These results suggest fibrosis in the SMG of affected patients is closely linked to the proliferation of fibroblasts following induction of IL-6 and WISP1 by inflammatory cytokines. The Th2 cytokines TSLP and IL-33 are also upregulated in affected SMG, and thus may cause chronic inflammation and IgG4 accumulation.

Guanylate binding protein-1-mediated epithelial barrier in human salivary gland duct epithelium.

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-1ß, TNFα and the growth factor TGF-ß. Treatment with IFNγ, IL-1ß, 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.

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.

The role of transcriptional factor p63 in regulation of epithelial barrier and ciliogenesis of human nasal epithelial cells.

Disruption of nasal epithelial tight junctions (TJs) and ciliary dysfunction are found in patients with chronic rhinosinusitis (CRS) and nasal polyps (NPs), along with an increase of p63-positive basal cells and histone deacetylase (HDAC) activity. To investigate these mechanisms, primary cultures of HNECs transfected with human telomerase reverse transcriptase (hTERT-HNECs) were transfected with siRNAs of TAp63 and ΔNp63, treated with the NF-kB inhibitor curucumin and inhibitors of HDACs, and infected with respiratory syncytial virus (RSV). In TERT-HNECs, knockdown of p63 by siRNAs of TAp63 and ΔNp63, induced claudin-1 and -4 with Sp1 activity and enhanced barrier and fence functions. The knockdown of p63 enhanced the number of microvilli with the presence of cilia-like structures. Treatment with curcumin and inhibitors of HDACs, or infection with RSV prevented expression of p63 with an increase of claudin-4 and the number of microvilli. The knockdown or downregulation of p63 inhibited phospho-p38MAPK, and the p38MAPK inhibitor downregulated p63 and upregulated the barrier function. Thus, epithelial barrier and ciliogenesis of nasal epithelium are regulated in a p63-negative manner in normal and upper airway diseases. Understanding of the regulation of p63/p38 MAPK/NF-κB may be important in the therapy for airway allergy and its drug delivery system.

Loss of tricellular tight junction protein LSR promotes cell invasion and migration via upregulation of TEAD1/AREG in human endometrial cancer.

Lipolysis-stimulated lipoprotein receptor (LSR) is a unique molecule of tricellular contacts of normal and cancer cells. We investigated how the loss of LSR induced cell migration, invasion and proliferation in endometrial cancer cell line Sawano. mRNAs of amphiregulin (AREG) and TEA domain family member 1 (TEAD1) were markedly upregulated by siRNA-LSR. In endometrial cancer tissues, downregulation of LSR and upregulation of AREG were observed together with malignancy, and Yes-associated protein (YAP) was present in the nuclei. siRNA-AREG prevented the cell migration and invasion induced by siRNA-LSR, whereas treatment with AREG induced cell migration and invasion. LSR was colocalized with TRIC, angiomotin (AMOT), Merlin and phosphorylated YAP (pYAP). siRNA-LSR increased expression of pYAP and decreased that of AMOT and Merlin. siRNA-YAP prevented expression of the mRNAs of AREG and TEAD1, and the cell migration and invasion induced by siRNA-LSR. Treatment with dobutamine and 2-deoxy-D-glucose and glucose starvation induced the pYAP expression and prevented the cell migration and invasion induced by siRNA-LSR. siRNA-AMOT decreased the Merlin expression and prevented the cell migration and invasion induced by siRNA-LSR. The loss of LSR promoted cell invasion and migration via upregulation of TEAD1/AREG dependent on YAP/pYAP and AMOT/Merlin in human endometrial cancer cells.

The Behavior and Role of Lipolysis-stimulated Lipoprotein Receptor, a Component of Tricellular Tight Junctions, in Head and Neck Squamous Cell Carcinomas.

BACKGROUND/AIM: Lipolysis-stimulated lipoprotein receptor (LSR) knockdown has also been reported to increase the motility and invasiveness of certain cancer cells. Here, we describe, for the first time, the behavior and role of LSR in head and neck squamous cell carcinoma (HNSCC) in vivo and in vitro. MATERIALS AND METHODS: Samples of HNSCC, normal palatine tonsils, the pharynx carcinoma cell line Detroit562 and primary cultured HNSCC were characterized by immunostaining, western blot, real-time polymerase chain reaction (PCR), Matrigel invasion and proliferation assays. RESULTS: Protein and mRNA of LSR were strongly expressed, as well as claudin-1 in HNSCC tissues than in normal tissues, especially in invasive tissues. Knock-down of LSR and claudin-1 (CLDN-1), but not tricellulin (TRIC) by siRNAs, markedly induced invasiveness of Detroit562 cells and primary cultured HNSCC. LSR inhibited the development and progression of HNSCC. CONCLUSION: LSR is a potential target for new forms of head and neck cancer therapy.

Accessory parotid gland tumors: A series of 4 cases.

Accessory parotid gland tumors are clinically rare, and their management remains unclear. In this article, we describe our experience with 4 patients-2 males and 2 females, aged 13 to 66 years-who were diagnosed with an accessory parotid gland tumor. All patients presented with an asymptomatic midcheek swelling, and all underwent fine-needle aspiration biopsy, ultrasonography, computed tomography, and magnetic resonance imaging. A standard parotidectomy was performed on all patients. Postoperatively, 2 patients were found to have a malignant tumor, while the other 2 had a pleomorphic adenoma. No patient experienced any obvious facial nerve injuries postoperatively, and no recurrences were observed. We discuss the preoperative evaluation, treatment, and prognosis of these tumors, and we briefly describe the literature. The first choice of treatment for accessory parotid gland tumors is surgical resection. In our experience, a standard parotidectomy approach is safe and cosmetically appealing.

Clarithromycin prevents human respiratory syncytial virus-induced airway epithelial responses by modulating activation of interferon regulatory factor-3.

Macrolide antibiotics exert immunomodulatory activity by reducing pro-inflammatory cytokine production by airway epithelial cells, fibroblasts, vascular endothelial cells, and immune cells. However, the underlying mechanism of action remains unclear. Here, we examined the effect of clarithromycin (CAM) on pro-inflammatory cytokine production, including interferons (IFNs), by primary human nasal epithelial cells and lung epithelial cell lines (A549 and BEAS-2B cells) after stimulation by Toll-like receptor (TLR) and RIG-I-like receptor (RLR) agonists and after infection by human respiratory syncytial virus (RSV). CAM treatment led to a significant reduction in poly I:C- and RSV-mediated IL-8, CCL5, IFN-ß and -λ production. Furthermore, IFN-ß promoter activity (activated by poly I:C and RSV infection) was significantly reduced after treatment with CAM. CAM also inhibited IRF-3 dimerization and subsequent translocation to the nucleus. We conclude that CAM acts a crucial modulator of the innate immune response, particularly IFN production, by modulating IRF-3 dimerization and subsequent translocation to the nucleus of airway epithelial cells. This newly identified immunomodulatory action of CAM will facilitate the discovery of new macrolides with an anti-inflammatory role.

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 ß-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, ß-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.

Junctional Adhesion Molecule-A in Head and Neck Squamous Cell Carcinoma.

Junctional adhesion molecule-A (JAM-A), which belongs to the IgG superfamily, is one of the tight junction molecules. JAM-A is dysregulated in various cancers and is closely associated with the invasion and metastasis of cancers such as breast cancer, lung cancer and pancreatic cancer. In the present study, we found a high expression of JAM-A in head and neck squamous cell carcinoma (HNSCC) as well as ß-catenin in immunohistochemistry. The expression of JAM-A and ß-catenin was of a low level in differentiation-induced cancer pearl regions of HNSCC. Real-time PCR showed the high expression of JAM-A mRNA through all differentiated stages (well, moderate, poor) of HNSCC. When we performed ELISA using the serum of HNSCC patients to measure plasma-soluble JAM-A, it was found to be higher in HNSCC patients than healthy subjects. These results indicate that JAM-A is one of the malignancy markers of HNSCC as well as ß-catenin in histopathology, and the plasma-soluble JAM-A may contribute to a serum diagnosis of HNSCC. JAM-A is a promising molecular target for diagnosis and therapy in HNSCC.

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

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.

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.

Irsogladine maleate regulates gap junctional intercellular communication-dependent epithelial barrier in human nasal epithelial cells.

The airway epithelium of the human nasal mucosa acts as the first physical barrier that protects against inhaled substances and pathogens. Irsogladine maleate (IM) is an enhancer of gastric mucosal protective factors via upregulation of gap junctional intercellular communication (GJIC). GJIC is thought to participate in the formation of functional tight junctions. However, the effects of IM on GJIC and the epithelial barrier in human nasal epithelial cells (HNECs) remain unknown. To investigate the effects of IM on GJIC and the tight junctional barrier in HNECs, primary cultures of HNECs transfected with human telomerase reverse transcriptase (hTERT-HNECs) were treated with IM and the GJIC inhibitors oleamide and 18ß-GA. Some cells were pretreated with IM before treatment with TLR3 ligand poly(I:C) to examine whether IM prevented the changes via TLR3-mediated signal pathways. In hTERT-HNECs, GJIC blockers reduced the expression of tight junction molecules claudin-1, -4, -7, occludin, tricellulin, and JAM-A. IM induced GJIC activity and enhanced the expression of claudin-1, -4, and JAM-A at the protein and mRNA levels with an increase of barrier function. GJIC blockers prevented the increase of the tight junction proteins induced by IM. Furthermore, IM prevented the reduction of JAM-A but not induction of IL-8 and TNF-α induced by poly(I:C). In conclusion, IM can maintain the GJIC-dependent tight junctional barrier via regulation of GJIC in upper airway nasal epithelium. Therefore, it is possible that IM may be useful as a nasal spray to prevent the disruption of the epithelial barrier by viral infections and exposure to allergens in human nasal mucosa.