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The stratified squamous epithelium has a multilayer structure formed by the differentiation of the keratinized epithelium, which covers the skin and oral mucosa. The epithelium plays a central role in regulating the interactions between the immune system and pathogens. The tight junction (TJ) barrier, which is composed of adhesion molecules called claudins (CLDN), is critical for the homeostasis of the skin and oral mucosa. Furthermore, the crucial roles of vitamin D3 (VD3) in the pathogeneses of skin and oral mucosal disease have been suggested. The aim of this in vitro study was to observe the correlations between the integrity of the keratinocyte population and the expression levels of CLDN1 and CLDN4 in gingival epithelial cells, stimulated with VD3. CLDN 1 and 4 expression levels were down and upregulated, respectively, in the cells stimulated with VD3. Additionally, transepithelial electrical resistance (TEER) levels were increased in the stimulated cells when compared to the controls. These findings indicate that CLDN 4 may play a more important role in the TJ barrier than CLDN 1. Hence, the therapeutic effect of VD3 in skin and oral diseases may be regulated by the increase in the expression of CLDN 4.

Immunohistochemical microarray comprising 80 patients with esophageal squamous cell carcinoma (ESCC) and discovered that the expression of CLDN1 and CLDN4 were significantly higher in cancer tissues compared to para-cancerous tissues. Furthermore, CLDN4 significantly affected the overall survival of cancer patients. When two ESCC cell lines (TE1, KYSE410) were exposed to hypoxia (0.1% O2), CLDN1/4 was shown to influence the occurrence and development of esophageal cancer. Compared with the control culture group, the cancer cells cultured under hypoxic conditions exhibited obvious changes in CLDN1 and CLDN4 expression at both the mRNA and protein levels. Through genetic intervention and Chip, we found that HIF-1α could directly regulate the expression of CLDN1 and CLDN4 in cancer cells. Hypoxia can affect the proliferation and apoptosis of cancer cells by regulating the PI3K-Akt-mTOR pathway. Molecular analysis further revealed that CLDN1 and CLDN4 can participate in the regulation process and had a feedback regulatory effect on HIF-1α expression in cancer cells. In vitro cellular experiments and vivo experiments in nude mice further revealed that changes in CLDN4 expression in cancer cells could affect the proliferation of cancer cells via regulation of Rho GTP and p-JNK pathway. Whether CLDN4 can be target for the treatment of ESCC needs further research.

Intestinal oxidative stress produces pro-inflammatory cytokines, which increase tight junction (TJ) permeability, leading to intestinal and systemic inflammation. Cystine (Cys2) is a substrate of glutathione (GSH) and inhibits inflammation, however, it is unclear whether Cys2 locally improves intestinal barrier dysfunction. Thus, we investigated the local effects of Cys2 on oxidative stress-induced TJ permeability and intestinal inflammatory responses. Caco-2 cells were cultured in a Cys2-supplemented medium for 24 h and then treated with H2O2 for 2 h. We assessed TJ permeability by measuring transepithelial electrical resistance and the paracellular flux of fluorescein isothiocyanate–dextran 4 kDa. We measured the concentration of Cys2 and GSH after Cys2 pretreatment. The mRNA expression of pro-inflammatory cytokines was assessed. In addition, the levels of TJ proteins were assessed by measuring the expression of TJ proteins in the whole cells and the ratio of TJ proteins in the detergent-insoluble fractions to soluble fractions (IS/S ratio). Cys2 treatment reduced H2O2-induced TJ permeability. Cys2 did not change the expression of TJ proteins in the whole cells, however, suppressed the IS/S ratio of claudin-4. Intercellular levels of Cys2 and GSH significantly increased in cells treated with Cys2. Cys2 treatment suppressed the mRNA expression of pro-inflammatory cytokines, and the mRNA levels were significantly correlated with TJ permeability. In conclusion, Cys2 treatment locally reduced oxidative stress-induced intestinal barrier dysfunction possively due to the mitigation of claudin-4 dislocalization. Furthermore, the effect of Cys2 on the improvement of intestinal barrier function is related to the local suppression of oxidative stress-induced pro-inflammatory responses.

Background Ovarian cancer has the highest mortality amongst all gynaecological malignancies, with around two-thirds of patients diagnosed with advanced disease due to late presentation. Furthermore, around 90% of patients develop recurrence and eventually become chemoresistant. Therefore, there is a high demand to identify biomarkers specific to this disease for screening for early detection, as well as new therapeutic targets. Tight junctions (TJs) regulate paracellular permeability and are vital in establishing epithelial cell polarity. One hallmark of tumorigenesis is the loss of TJs, with loss of cell-to-cell adhesion. Claudins are integral TJ membrane proteins, which have been found to play a critical role in maintaining the TJ’s barrier function. Furthermore, claudin-3 (CLDN3) and claudin-4 (CLDN4) are overexpressed in ovarian cancer. This article aims to explore the biological role of CLDN3 and CLDN4 and their potential in different aspects of the management of ovarian cancer. Main body CLDN3 and CLDN4 have been shown to be effective markers for the early detection of ovarian cancer. Whilst there is difficulty in screening for both claudins in serum, their assessment by gene expression analysis and immunohistochemical methods shows promising potential as diagnostic and prognostic biomarkers for ovarian cancer. The localisation and overexpression of claudins, such as CLDN3, have been shown to correlate with poorer survival outcomes. The added value of combining claudins with other markers such as CA125 for diagnosis has also been highlighted. Therapeutically, CLDN3 and more so CLDN4 have been shown to be effective targets of Clostridium perfringens enterotoxin (CPE). Interestingly, CPE has also been shown to resensitise chemoresistant tumours to therapy. Conclusions This review presents the diagnostic and prognostic potential of CLDN3 and CLDN4 and their emerging role as therapeutic targets in ovarian cancer. Clinical trials are required to validate the promising results of the in vitro and in vivo studies for CLDN3 and CLDN4, possibly adding onto current ovarian cancer management.

In this study we designed a claudin 4-directed dual photodynamic and photothermal system, in which a 30-amino acid claudin 4-binding peptide, Clostridium perfringens enterotoxin (CPE), was linked to a photodynamic agent chlorin e6 (Ce6) through a polyethylene glycol spacer (CPC) and anchored onto reduced graphene oxide (rGO) nanosheets to form CPC/rGO nanosheets. For comparison, a conjugate of polyethylene glycol and Ce6 (PC) was anchored onto the rGO nanosheets to generate PC/rGO. Both PC and CPC generated reactive oxygen species upon irradiation at 660 nm. Application of CPC/rGO to claudin 4-overexpressing U87 glioblastoma cells in vitro resulted in a significantly higher cellular uptake compared to application of PC/rGO. Upon irradiation at 660 and 808 nm, the CPC/rGO-treated U87 cells generated significantly higher reactive oxygen species and caused significantly higher temperature increase, and showed most potent anticancer effect compared to the other groups. Taken together, these results suggest that CPC/rGO is potentially useful as a tumor-specific combined phototherapy.

We previously reported that TU-100 suppresses irinotecan hydrochloride (CPT-11)-induced inflammatory cytokines and apoptosis. However, the mechanism underlying this effect has not been fully elucidated. The aim of this study was to further clarify the mechanism of CPT-11-induced bacterial translocation (BT) and the effect of TU-100 on BT. Cell cytotoxicity was assessed in vitro by a WST-8 assay. For the in vivo experiments, rats were randomly divided into 3 groups: the control group, the CPT-11 group (250 mg/kg i.p. for 2 days), and the CPT-11 and TU-100 co-treated group (1000 mg/kg, p.o. for 5 days). All of the rats were sacrificed on day 6 and their tissues were collected. CPT-11 and TU-100 co-treatment improved CPT-11 the related cytotoxicity in vitro. All CPT-11-treated rats developed different grades of diarrhea and BT was observed in 80% of the rats. CPT-11 caused a significant increase in the expression of TLR4, IL-6, TNF-α, IL-1β and caspase-3 mRNAs in the large intestine. The expression of tight junction (TJ) marker mRNAs (occludin, claudin-1 and 4, and ZO-1) was significantly decreased in comparison to the control group. TU-100 co-treatment significantly reversed diarrhea, BT, and the expression of TLR2, IL-6, TNF-α, IL-1β and caspase-3, and improved the expression of occludin, claudin-4 and ZO-1. TU-100 can suppress the adverse effects associated with CPT-11 and improve the function of the TJ. It is possible that this occurs through the TLR pathway.

Abnormal expression of claudin (Cldn), the main constituent of tight junctions, may play a crucial role in carcinogenesis. To elucidate these abnormalities of tight junctions in lung adenocarcinoma during carcinogenesis, we examined immunohistochemical expressions of Cldn4 and Cldn7 in human lung resection materials. Lung resection specimens from 86 patients were studied, including 16 atypical adenomatous hyperplasia (AAH), 19 adenocarcinoma in situ (AIS), 32 invasive adenocarcinoma (ADC), 5 AIS with AAH, 2 ADC with AAH, 10 ADC with AIS, and 2 ADC with AIS and AAH. The immunohistochemical staining (IHC) score was defined for both the extent and intensity of staining. IHC score for Cldn4 in AIS and ADC was significantly higher than that in alveolar epithelium (AE) and AAH (p

Claudins constitute a family of at least 27 proteins with four transmembrane domains, and play a pivotal role in maintaining tight-junctions seals in diverse epithelial tissues. The expression of claudin-4 often changes in intestinal tissues of inflammatory bowel disease and various human cancers. Therefore, claudin-4 is a promising target for treatment of these diseases. In our previous study, we established a reporter cell line to monitor claudin-4 expression on the basis of a functional claudin-4 promoter. Using this cell line, we have performed a cell-based screen of a library containing 2642 biologically active small-molecule compounds to identify modulators of claudin-4 expression. The screen identified 24 potential modulators of the claudin-4 promoter activity. Fourteen of these compounds (12 of them novel) induced endogenous claudin-4 expression. The identified compounds might serve as lead compounds targeting aberrant gene expression in inflammatory bowel disease.

Thousands of genes have been well demonstrated to play important roles in cancer progression. As genes do not function in isolation, they can be grouped into “networks” based on their interactions. In this study, we discover a network regulating Claudin-4 in gastric cancer. We observe that Claudin-4 is up-regulated in gastric cancer and is associated with poor prognosis. Claudin-4 reinforce proliferation, invasion, and EMT in AGS, HGC-27, and SGC-7901 cells, which could be reversed by miR-596 and miR-3620-3p. In addition, lncRNA-KRTAP5-AS1 and lncRNA-TUBB2A could act as competing endogenous RNAs to affect the function of Claudin-4. Our results suggest that non-coding RNAs play important roles in the regulatory network of Claudin-4. As such, non-coding RNAs should be considered as potential biomarkers and therapeutic targets against gastric cancer., Non-coding RNAs can modify the expression of proteins in cancer networks. Here the authors reveal a regulatory network in gastric cancer whereby claudin-4 expression is reduced by specific miRNAs, which are in turn bound by specific lncRNAs acting as competing endogenous RNAs (ceRNAs), resulting in increased claudin-4 expression.

Claudins constitute a group of principal proteins forming the tight junctional complex. The altered expression of selected claudins has been reported in several human cancers. The purpose of this study was to investigate the expression of claudin-1 and claudin-4 in oral squamous cell carcinoma (OSCC) and examine its relationship with patient clinical-pathologic features. Forty-five OSCC cases were enrolled. Patient clinical, pathologic and follow-up data were reviewed and the claudin-1 and claudin-4 expression was analyzed immunohistochemically. Positive claudin-1 and claudin-4 immunoreactivities were noted in 86.7 and 80 % of cases, respectively. The majority of cases showed the staining in less than 25 % of cancer cells. The increased claudin-1 expression was significantly associated with the high pathologic grade, the presence of microscopic perineural invasion, vascular invasion, nodal metastasis, and advanced clinical stage. No relationship between various clinico-pathologic parameters and differential claudin-4 expression was observed. Claudin-1 may play a role in OSCC progression and could serve as a prognostic marker of advanced disease.

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.

To research the effects of silencing transcription factor SNAI1 on the in vitro biological phenotypes of breast cancer cell line MCF-7, based on the gene sequence of SNAI1, we linked shRNA with the green fluorescent protein-expressing eukaryotic expression vector pGCsilencer™ U6/Neo/GFP, and transfected it into MCF-7 cells. The SNAI1 gene-silencing effect was authenticated by RT-PCR and immunofluorescence. We then examined the effect of gene silencing on the expression of epithelial and mesenchymal markers and on their biological phenotypes of the target cells. Finally, we explained that SNAI1 was bound to E-cadherin in MCF-7 cells by ChIP. Silencing SNAI1 upregulated the expression of epithelial markers claudin-4, claudin-7, and E-cadherin, while expression of the mesenchymal marker matrix metalloproteinase-2 was downregulated. The capacity for proliferation, migration, and invasion was diminished. SNAI1 binds to the E-cadherin gene promoter and inhibits its transcription. We can conclude that silencing gene SNAI1 inhibits expression of properties that are associated with the malignant phenotype of MCF-7 cells and reverses the epithelial-mesenchymal transition process by regulating relevant target gene E-cadherin.

Methotrexate (MTX)-induced intestinal mucositis limits the use of the drug. We previously reported that MTX-dependent production of reactive oxygen species is an initiating signal leading to neutrophil migration and intestinal barrier dysfunction. Moreover, alterations of zonula occludens (ZO)-1, an integral component of tight junctions (TJs), contribute to its dysfunction. This study aimed to clarify the identity of inflammatory mediators in the intestine of MTX-treated rats and to evaluate MTX-stimulated alterations in the expression of TJ proteins other than ZO-1 (e.g., occludin and claudins). Male Wistar rats were administrated MTX (15 mg kg−1) orally once daily for 4 days. Tumor necrosis factor (TNF)-α, interleukin (IL)-1β, macrophage inflammatory protein (MIP)-2, cytokine-induced neutrophil chemoattractant-2, Toll-like receptor 4 (TLR4), and occludin were determined by real-time RT-PCR. Expression, distribution, and interactions of TJ proteins were evaluated by Western blotting, immunohistochemistry, and immunoprecipitation. MTX increased the mRNA levels of TNF-α, IL-1β, MIP-2, and TLR4 in the small intestine, as well as the protein expression of claudin-2. Increased claudin-2 and decreased claudin-4 immunostaining were also observed. Occludin mRNA levels were significantly diminished by MTX administration, whereas occludin protein levels and the interaction between ZO-1 and occludin were unaltered; however, the interaction between ZO-1 and claudin-4 was significantly compromised. These results indicate that elevated levels of inflammatory cytokines and chemokines in the small intestine of MTX-treated rats may contribute to the inhibition of ZO-1/claudin-4 binding, and that inhibition of ZO-1/claudin-4 binding may in turn lead to a reduction in claudin-4 expression.

Background Claudin-4 is a transmembrane protein expressed at high levels in the majority of epithelial ovarian tumors, irrespective of subtype, and has been associated with tumor cells that are both chemoresistant and highly mobile. The objective of this study was to determine the functional role that claudin-4 plays in apoptosis resistance and migration as well as the therapeutic utility of targeting claudin-4 activity with a small mimic peptide. Methods We examined claudin-4 activity in human ovarian tumor cell lines (SKOV3, OVCAR3, PEO4) using in vitro caspase and scratch assays as well as an in vivo mouse model of ovarian cancer. Claudin-4 activity was disrupted by treating cells with a small peptide that mimics the DFYNP sequence in the second extracellular loop of claudin-4. Claudin-4 expression was also altered using shRNA-mediated gene silencing. Results Both the disruption of claudin-4 activity and the loss of claudin-4 expression significantly increased tumor cell caspase-3 activation (4 to 10-fold, respectively) in response to the apoptotic inducer staurosporine and reduced tumor cell migration by 50 %. The mimic peptide had no effect on cells that lacked claudin-4 expression. Female athymic nude mice bearing ZsGreen-PEO4 ovarian tumors showed a significant decrease in ovarian tumor burden, due to increased apoptosis, after treatment with intraperitoneal injections of 4 mg/kg mimic peptide every 48 h for three weeks, compared to control peptide treated mice. Conclusion Claudin-4 functionally contributes to both ovarian tumor cell apoptosis resistance and migration and targeting extracellular loop interactions of claudin-4 may have therapeutic implications for reducing ovarian tumor burden.

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.

Bacterial toxins are known to be effective for cancer therapy. Clostridium perfringens enterotoxin (CPE) is produced by the bacterial Clostridium type A strain. The transmembrane proteins claudin-3 and -4, often overexpressed in numerous human epithelial tumors (for example, colon, breast, pancreas, prostate and ovarian), are the targeted receptors for CPE. CPE binding to them triggers formation of membrane pore complexes leading to rapid cell death. In this study, we aimed at selective tumor cell killing by CPE gene transfer. We generated expression vectors bearing the bacterial wild-type CPE cDNA (wtCPE) or translation-optimized CPE (optCPE) cDNA for in vitro and in vivo gene therapy of claudin-3- and -4-overexpressing tumors. The CPE expression analysis at messenger RNA and protein level revealed more efficient expression of optCPE compared with wtCPE. Expression of optCPE showed rapid cytotoxic activity, hightened by CPE release as bystander effect. Cytotoxicity of up to 100% was observed 72 h after gene transfer and is restricted to claudin-3-and -4-expressing tumor lines. MCF-7 and HCT116 cells with high claudin-4 expression showed dramatic sensitivity toward CPE toxicity. The claudin-negative melanoma line SKMel-5, however, was insensitive toward CPE gene transfer. The non-viral intratumoral in vivo gene transfer of optCPE led to reduced tumor growth in MCF-7 and HCT116 tumor-bearing mice compared with the vector-transfected control groups. This novel approach demonstrates that CPE gene transfer can be employed for a targeted suicide gene therapy of claudin-3- and -4-overexpressing tumors, leading to the rapid and efficient tumor cell killing in vitro and in vivo.

Tight junctions (TJs) are the most apical junctional complexes and restrict the fluid flux through the paracellular pathway. In the mammary glands, the tightness of TJs occurs shortly after parturition to prevent the leakage of milk components from the lumen and the loosening of TJs is induced immediately after weaning. Claudins are transmembrane proteins, and their composition at the apical-most regions determines the permeability of TJs. In this study, we investigated the localization and expression patterns of claudin-3 and -4 in the mammary glands around the lactation period because it is unclear how claudins construct mammary TJs in the apical-most regions. Our results showed that claudin-3 and -4 change not only their level of expression but also their localization in the processes of parturition, lactation, and weaning. Claudin-3 was concentrated in the apical-most regions during lactation, whereas claudin-4 gradually decreased at the beginning of lactation and increased drastically immediately after weaning. The qualitative change of claudin-3 was also identified by western blotting analysis as an additional band around the lactation period. In addition, parts of the mammary epithelial cells showed intensive positive reactions to claudin-4 in the lateral membrane and cytoplasm after weaning, concurrently with the involution mammary glands. These results indicate that claudin-3 in the apical-most regions maintains the impermeable TJs during lactation, and claudin-4 contributes to the permeability changes of TJs immediately after parturition and weaning.

Identification of prognostic markers would be useful in the clinical management of patients with pancreatic ductal adenocarcinoma (PDAC). The clinical relevance of claudin-4 (CLDN4), recently identified as overexpressed in PDAC, is unknown. Using quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR), we analyzed CLDN4 mRNA expression in a panel of 9 pancreatic cancer cell lines and formalin-fixed paraffin-embedded (FFPE) tissues from 100 patients with PDAC. The CLDN4 expression levels were then correlated with clinicopathological variables and patient outcome. We also performed immunohistochemical analysis in 20 FFPE samples of PDAC to investigate the expression of CLDN4 protein. Increased expression of CLDN4 was confirmed in all the pancreatic cancer cell lines tested compared with normal ductal epithelial cells and fibroblasts. We found that low expression of CLDN4 was significantly associated with shorter survival in patients with PDAC (hazard ratio; 1.362, 95% confidence interval; 1.011–1.873, P = 0.0419). Patients with high CLDN4 expression survived longer for a median of 63.0 months, compared with 14.7 months in patients with low CLDN4 expression (P = 0.0067). In immunohistochemical analysis, the level of CLDN4 mRNA expression was significantly correlated with the expression of CLDN4 protein (P = 0.0168). Increased expression of CLDN4 mRNA predicts better prognosis in PDAC.

The precise characterization of post translational modifications (PTMs) is important for the understanding of protein regulatory mechanisms and their role in disease. However, experimental studies on PTMs, especially with multifunctional proteins are difficult to follow and investigate. Bioinformatic tools are therefore helpful in predicting key protein modifications. To study the role of PTMs in claudin proteins, specifically claudin-1, -3 and -4 in the onset or progression of human cancers, we performed an in silico study of various PTMs and investigated their interplay. Given that the activity of claudins is known to be influenced by two types of PTMs, specifically palmitoylation and kinase- dependent phosphorylation, we predicted two conserved regions in the topological domains of claudin-1, -3 and -4 as potential palmitoylation sites. Furthermore, conserved phosphorylation residues, which may be targets for kinases and can alter claudin's ability to maintain the integrity of tight junctions, were identified. To our knowledge, this is the first report to suggest O-glycosylation of claudin proteins, as well as a potential novel interplay between phosphorylation and O-glycosylation at Yin Yang sites. Thus, our findings may facilitate the production of anti-cancer drugs, and suggest that novel therapeutic strategies should target post translational events.

Testosterone and inflammation have been linked to the development of common age-associated diseases affecting the prostate gland including prostate cancer, prostatitis, and benign prostatic hypertrophy. We hypothesized that testosterone regulates components of prostate tight junctions which serve as a barrier to inflammation, thus providing a connection between age- and treatment-associated testosterone declines and prostatic pathology. We examined the expression and distribution of tight junction proteins in prostate biospecimens from mouse models and a clinical study of chemical castration, using transcript profiling, immunohistochemistry and electron microscopy. We determined that low serum testosterone is associated with reduced transcript and protein levels of Claudin 4 and Claudin 8, resulting in defective tight junction ultrastructure in benign prostate glands. Expression of Claudin 4 and Claudin 8 was negatively correlated with the mononuclear inflammatory infiltrate caused by testosterone deprivation. Testosterone suppression also induced an auto-immune humoral response directed toward prostatic proteins. Testosterone supplementation in castrate mice resulted in re-expression of tight junction components in prostate epithelium and significantly reduced prostate inflammatory cell numbers. These data demonstrate that tight junction architecture in the prostate is related to changes in serum testosterone levels, and identify an androgen-regulated mechanism that potentially contributes to the development of prostate inflammation and consequent pathology.

Claudins are 22-27 kDa sized adhesion molecules that constitute tight junctions. Their expression levels are often tissue-specific, and their altered degrees of expression have been reported in a variety of cancers. In addition, the prognostic significance of claudin expression has been implicated in various human cancers. However, the prognostic significance of claudin-4 expression in esophageal squamous cell carcinoma (ESCC) remains to be clarified.We investigated the prognostic significance of claudin-4 expression in 164 cases of ESCC using immunohistochemisty. We also evaluated claudin-4 mRNA expression levels using quantitative real-time reverse transcription polymerase chain reaction (RT-PCR) and analyzed its specific promoter methylation status using quantitative methylation-specific PCR.According to clinicopathological parameters, low claudin-4 expression was found to be significantly associated with histological differentiation (P = 0.003), invasion depth (P = 0.002), and lymph node metastasis (P = 0.024). Low claudin-4 expression showed unfavorable influences on disease-free survival (P = 0.0115) and overall survival (OS) (P = 0.0009). In multivariate analysis, low claudin-4 expression was an independent predictor of poor OS (P = 0.007). Claudin-4 mRNA levels assessed using real-time RT-PCR were consistent with the protein levels determined using immunohistochemistry. Furthermore, this study demonstrates that loss of claudin-4 is associated with promoter hypermethylation.Our study indicates that claudin-4 expression is deregulated in ESCC, implying its potential use as a prognostic biomarker in ESCC.

Duodenal biopsy is an important tool to diagnose coeliac disease (CD); however, the most reliable location of biopsy site is still questionable. Claudins (CLDNs), members of a large family of adherent junction proteins, show characteristic expression pattern in inflammatory disorders; nevertheless, CLDN expression in CD is unknown. This is a comparative study to examine the CLDN 2, 3 and 4 expressions in proximal and distal part of duodenum in children with CD and in controls. Thirty-three children with newly diagnosed CD were enrolled. Fourteen healthy children served as controls. Biopsies from proximal and distal part of duodenum were taken for routine histological analysis. Immunohistochemistry were used to detect CD3+ intraepithelial lymphocytes and CLDN 2, 3 and 4 protein expressions. Macroscopic picture, routine histology and Marsh grade depicted no differences between biopsies taken from proximal or distal part of duodenum. However, CLDN 2 expression was significantly increased in severe form of coeliac disease in bulb and in distal duodenum, and in distal part of non-severe coeliac patients, in comparison to controls. Similar association was found concerning CLDN 3 expression. Expression of CLDN 4 was similar in all groups studied. Both proximal and distal mucosal duodenal biopsies are suitable for diagnosing villous atrophy in patients with CD. Increased expressions of CLDN 2 and 3 suggest structural changes of tight junction in coeliac disease which may be, at least in part, responsible for increased permeability and proliferation observed in coeliac disease.

Junctional epithelium, a nonkeratinized stratified epithelium, extends apically in apposition to the surface of the enamel to form a seal between the epithelium and the tooth. Desmosomes and gap junctions adhere to the junctional epithelium through cell-cell contact, but no evidence of tight junctions has been found. Recently, tight junction hallmark proteins and tight junction-related structures have been identified in stratified squamous epithelium. The present study examined whether tight junction proteins were expressed in the junctional epithelium. We used immunohistochemical techniques to observe expression of claudin-1, -4, -5, -7, and occludin in porcine gingival junctional epithelium. Claudin-4 exhibited immunoreactivity in the intercellular spaces of all layers of the oral epithelium and the junctional epithelium. Stronger expression was observed in junctional epithelial cells adjacent to the inner and outer basal laminae than in the inner cell layers. Immunohistochemical positivity for claudin-7 was clearly observed in the junctional epithelium, but only a faint positivity was observed in the basal layer of the oral epithelium. No immunohistochemical positivity for claudin-1, -5, or occludin was observed in the junctional epithelium. RT-PCR assay confirmed expression of porcine claudin-4 and -7 mRNAs in the junctional epithelium. These findings indicate that claudin-4 and -7 may play a role in the junctional epithelium even in the absence of tight junctions.

Tight junction (TJ) proteins play a critical role in cellular adhesion, glandular differentiation, and cellular proliferation. The function of these proteins is compromised in a number of intestinal diseases, including ulcerative colitis that has an increased incidence for colorectal carcinoma (CAC). The aim of this study was to determine the expression of TJ proteins, claudin-1-4, occludin, ZO-1, and the adherens junction (AJ) protein beta-catenin in CAC.Sixteen colectomy specimens with CAC, adjoining intraepithelial neoplasia, and normal mucosa were studied by immunofluorescence. A semiquantitative evaluation of all investigated proteins was performed by scoring the staining intensity, and the TJ and AJ protein expression in neoplastic cells was compared to normal and intraepithelial neoplastic colonic mucosa.Using an intensity scoring system, mucosa of crypts and surfaces of CAC exhibited significantly elevated expression levels of claudin-1, claudin-3, claudin-4, and beta-catenin compared to intraepithelial neoplasia and normal mucosa (p0.05). These data were confirmed by a comparative score. The expression of claudin-2, occludin, and ZO-1 showed no differences between the groups.TJ proteins claudin-1, claudin-3, claudin-4, and the AJ protein beta-catenin are overexpressed in CAC. This suggests that these proteins may become potential markers and targets in CAC.

Although Smad signalling is known to play a tumour suppressor role, it has been shown to play a prometastatic function also in breast cancer and melanoma metastasis to bone. In contrast, mutation or reduced level of Smad4 in colorectal cancer is directly correlated to poor survival and increased metastasis. However, the functional role of Smad signalling in metastasis of colorectal cancer has not been elucidated. We previously reported that overexpression of Smad7 in colon adenocarcinoma (FET) cells induces tumorigenicity by blocking TGF-beta-induced growth inhibition and apoptosis. Here, we have observed that abrogation of Smad signalling by Smad7 induces liver metastasis in a splenic injection model. Polymerase chain reaction with genomic DNA from liver metastases indicates that cells expressing Smad7 migrated to the liver. Increased expression of TGF-beta type II receptor in liver metastases is associated with phosphorylation and nuclear accumulation of Smad2. Immunohistochemical analyses have suggested poorly differentiated spindle cell morphology and higher cell proliferation in Smad7-induced liver metastases. Interestingly, we have observed increased expression and junctional staining of Claudin-1, Claudin-4 and E-cadherin in liver metastases. Therefore, this report demonstrates, for the first time, that blockade of TGF-beta/Smad pathway in colon cancer cells induces metastasis, thus supporting an important role of Smad signalling in inhibiting colon cancer metastasis.

A fusion protein expression system is described that allows for production of eukaryotic integral membrane proteins in Escherichia coli (E. coli). The eukaryotic membrane protein targets are fused to the C terminus of the highly expressed E. coli inner membrane protein, GlpF (the glycerol-conducting channel protein). The generic utility of this system for heterologous membrane-protein expression is demonstrated by the expression and insertion into the E. coli cell membrane of the human membrane proteins: occludin, claudin 4, duodenal ferric reductase and a J-type inwardly rectifying potassium channel. The proteins are produced with C-terminal hexahistidine tags (to permit purification of the expressed fusion proteins using immobilized metal affinity chromatography) and a peptidase cleavage site (to allow recovery of the unfused eukaryotic protein).

Inhalation of hyperosmotic solutions (salt, mannitol) has been used in the treatment of patients with cystic fibrosis or asthma, but the mechanism behind the effect of hyperosmotic solutions is unclear. The relation between osmolarity and permeability changes was examined in an airway cell line by the addition of NaCl, NaBr, LiCl, mannitol, or xylitol (295-700 mOsm). Transepithelial resistance was measured as an indicator of the tightness of the cultures. Cell-cell contacts and morphology were investigated by immunofluorescence and by transmission electron microscopy, with lanthanum nitrate added to the luminal side of the epithelium to investigate tight junction permeability. The electrolyte solutions caused a significant decrease in transepithelial resistance from 450 mOsm upwards, when the hyperosmolar exposure was gradually increased from 295 to 700 mOsm; whereas the nonelectrolyte solutions caused a decrease in transepithelial resistance from 700 mOsm upwards. Old cultures reacted in a more rigid way compared to young cultures. Immuno-fluorescence pictures showed weaker staining for the proteins ZO-1, claudin-4, and plakoglobin in treated samples compared to the control. The ultrastructure revealed an increased number of open tight junctions as well as a disturbed morphology with increasing osmolarity, and electrolyte solutions opened a larger proportion of tight junctions than nonelectrolyte solutions. This study shows that hyperosmotic solutions cause the opening of tight junctions, which may increase the permeability of the paracellular pathway and result in increased transepithelial water transport.

Tight junctions are essential for the maintenance of epithelial cell polarity. We have previously established a system for the primary culture of salivary parotid acinar cells that retain their ability to generate new secretory granules and to secrete proteins in a signal-dependent manner. Because cell polarity and cell-cell adhesion are prerequisites for the formation of epithelial tissues, we have investigated the structure of the tight junctions in these cultures. We have found two types of cellular organization in the culture: monolayers and semi-spherical clusters. Electron microscopy has revealed tight junctions near the apical region of the lateral membranes between cells in the monolayers and cells at the surface of the clusters. The cells in the interior of the clusters also have tight junctions and are organized around a central lumen. These interior cells retain more secretory granules than the surface or monolayer cells, suggesting that they maintain their original character as acinar cells. The synthesis of claudin-4 increases during culture, although it is not detectable in the cells immediately after isolation from the glands. Immunofluorescence microscopy has shown that claudin-4 is synthesized in the monolayers and at the surface of the clusters, but not inside the clusters. Only claudin-3, which is present in the original acinar cells following isolation and in the intact gland, has been detected inside the clusters. These results suggest that differences in claudin expression are related to the three-dimensional structures of the cell cultures and reflect their ability to function as acinar cells.

Transfection of chicken vinculin into highly malignant neuroendocrine tumor cells, vasostatin-transformed (vaso-transformed) Bon cells which expressed low levels of vinculin protein, reversed their malignant behavior and restored expression of tumor suppressor genes. Conversely, small interfering RNA (siRNA)-mediated knockout of vinculin resulted in fast cell growth and augmentation of colony formation in wild-type cells. Moreover, expression of a tight junction protein, claudin 4 (CLD4), was found to be associated with vinculin expression. In the vaso-transformed Bon cells, CLD4 expression was reduced, whereas a significantly increased CLD4 expression was observed in the cells with vinculin overexpression. Furthermore, vinculin knockout brought about CLD4 downregulation in wild-type cells. However, vinculin and CLD4 expression was inversely correlated in neuroendocrine tumors, respectively. Based on these findings, we hypothesize that vinculin plays a role in growth regulation of neuroendocrine tumors. Further studies are necessary to analyze the relationship between the course of the disease, and vinculin and CLD4 expression in large tumor samples.

Background The current reports on the association of claudin-4 expression with gastric cancer outcome were inconsistent. Thus, we conducted a meta-analysis to assess the association of claudin-4 expression with the prognosis and clinical parameters more precisely. Methods Systematic searches on PubMed, Embase, and Cochrane Library prior to December 2014 were performed. The pooled hazard ratio (HR) with its 95 % confidence interval (95 %CI) was used to assess the prognostic value of claudin-4 expression with gastric cancer patients, and the pooled odds ratio (OR) with its 95 %CI was used to assess the association with clinical parameters. Results Nine studies with a total of 1265 gastric cancer patients were included. Overall, the pooled results showed that over-expression of claudin-4 was associated with a poor survival in gastric cancer patients (HR: 2.01, 95 % CI: 1.62–2.50). Over-expression of claudin-4 was also associated with advanced stage (OR: 1.96, 95 % CI: 1.08–3.56) and lymphoid node metastasis (OR: 1.72, 95 % CI: 1.05–2.81) of gastric cancer patients. No significant publication bias was found among the studies (P > 0.05). Conclusions This meta-analysis shows that over-expression of claudin-4 is associated with progress of gastric cancer and poor prognosis of gastric cancer patients.

The tight junction seal formed between epithelial cells varies among tissues in both tightness and ionic charge selectivity. We recently demonstrated that the extracellular domains of the claudin family of proteins are determinants of both characteristics, but in that study other unidentified domains in the claudins clearly contributed to their physiological potency. To investigate the importance of the cytoplasmic carboxyl-terminal domains in determining the degree to which a claudin can influence barrier properties, we constructed chimeras by exchanging the tails of claudin-2 and -4 and expressing them in MDCK II cells. Although swapping these domains had little effect on claudin localization, we found that the tail of claudin-2 could stabilize claudin-4, with a concomitant increase in both protein level and physiologic influence. This difference in stability was not an artifact of their chimeric structure, since metabolic radio-labeling experiments revealed that the half-life of endogenous claudin-2 is more than three times longer than claudin-4 (12 h and approximately 4 h respectively). Further, half-life was not affected by removing the carboxyl-terminal three amino acids, which form a PDZ-binding motif. The finding that cytoplasmic tails of claudins strongly influence stability reveals a potential mechanism by which cells can establish their tight junction protein composition and thus function.

Background Claudins are key integral proteins of the tight junction. Although they play an essential role in controlling paracellular diffusion in epithelia, increasing evidence supports a role for these proteins in non-barrier forming activities. To elucidate a potential function for claudins outside of their traditional role in tight junctions, subcellular localization of claudin-4 was determined in normal mammary epithelial cells as well as breast and ovarian cancer cell lines and the effects of a claudin mimic peptide on cell motility were determined. Results Immunofluorescence revealed that claudin-4 was localized along cellular projections. Using a fluorescent peptide that mimics a conserved sequence in the second extracellular loop of a set of claudin subtypes, that includes claudin-4, exposure of this loop to the extracellular environment was confirmed in non-polarized cells. This peptide inhibited cell motility when normal mammary epithelial cells as well as breast and ovarian tumor cells were subjected to a wound healing assay. Knockdown of claudin-4 also inhibited cell motility and the mimic peptide had no effect on motility in the claudin-4 knockdown cells. This effect on motility was seen when cells were grown on collagen, but not when cells were grown on non-physiological cell adhesive or fibronectin. Conclusion The second extracellular loop of claudins is able to interact with the extracellular environment to promote normal and tumor cell motility when it is not associated with tight junction structures.

Background Treatment of tumors with macromolecular toxins directed to cytoplasmic targets requires selective endocytosis followed by release of intact toxin from the endosomal/lysosomal compartment. The latter step remains a particular challenge. Claudins 3 and 4 are tight junction proteins that are over-expressed in many types of tumors. This study utilized the C-terminal 30 amino acid fragment of C. perfringens enterotoxin (CPE), which binds to claudins 3 and 4, to deliver a toxin in the form of recombinant gelonin (rGel) to the cytoplasm of the human ovarian carcinoma cell line 2008. Results CPE was fused to rGel at its N-terminal end via a flexible G4S linker. This CPE-G4S-rGel molecule was internalized into vesicles from which location it produced little cytotoxicity. To enhance release from the endosomal/lysosomal compartment a poly-arginine sequence (R9) was introduced between the CPE and the rGel. CPE-R9-rGel was 10-fold more cytotoxic but selectivity for claudin-expressing cells was lost. The addition of a poly-glutamic acid sequence (E9) through a G4S linker to R9-rGel (E9-G4S-R9-rGel) largely neutralized the non-selective cell membrane penetrating activity of the R9 motif. However, introduction of CPE to the E9-G4S-R9-rGel fusion protein (CPE-E9-G4S-R9-rGel) further reduced its cytotoxic effect. Treatment with the endosomolytic reagent chloroquine increased the cytotoxicity of CPE-E9-G4S-R9-rGel. Several types of linkers susceptible to cleavage by furin and endosomal cathepsin B were tested for their ability to enhance R9-rGel release but none of these modifications further enhanced the cytotoxicity of CPE-E9-G4S-R9-rGel. Conclusion We conclude that while a claudin-3 and -4 ligand serves to deliver rGel into 2008 cells the delivered molecules were entrapped in intracellular vesicles. Incorporation of R9 non-specifically increased rGel cytotoxicity and this effect could be masked by inclusion of an E9 sequence. However, the putative protease cleavable sequences tested were inadequate for release of R9-rGel from CPE-E9-G4S-R9-rGel.