Search

The tight junction protein claudin-7 is essential for tight junction function and intestinal homeostasis. Cldn7 deletion in mice leads to an inflammatory bowel disease-like phenotype exhibiting severe intestinal epithelial damage, weight loss, inflammation, mucosal ulcerations, and epithelial hyperplasia. Claudin-7 has also been shown to be involved in cancer metastasis and invasion. Here, we test our hypothesis that claudin-7 plays an important role in regulating colonic intestinal stem cell function. Conditional knockout of Cldn7 in the colon led to impaired epithelial cell differentiation, hyperproliferative epithelium, a decrease in active stem cells, and dramatically altered gene expression profiles. In 3D colonoid culture, claudin-7-deficient crypts were unable to survive and form spheroids, emphasizing the importance of claudin-7 in stem cell survival. Inhibition of the Hippo pathway or activation of Notch signaling partially rescued the defective stem cell behavior. Concurrent Notch activation and Hippo inhibition resulted in restored colonoid survival, growth, and differentiation to the level comparable to those of wild-type derived crypts. In this study, we highlight the essential role of claudin-7 in regulating Notch and Hippo signaling-dependent colonic stem cell functions, including survival, self-renewal, and differentiation. These new findings may shed light on potential avenues to explore for drug development in colorectal cancer., (© 2024 The Authors. Annals of the New York Academy of Sciences published by Wiley Periodicals LLC on behalf of The New York Academy of Sciences.)

Background & Aims: Claudin-7 (Cldn7) is a tight junction (TJ) membrane protein located at the apical TJ and basolateral side of intestinal epithelial cells. Deletion of Cldn7 by gene targeting leads to the inflammatory bowel disease-like phenotype in mice, which includes weight loss, diarrhea, mucosa ulceration, and severe intestinal epithelial damage. In this study, we test our hypothesis that Cldn7 plays a critical role in regulating intestinal crypt stem cell functions., Methods: Gene expression microarray, quantitative reverse-transcription polymerase chain reaction, in situ hybridization, histologic examinations, immunoblotting, 3-dimensional organoid culture, and various treatments to rescue Cldn7-deficient organoid defects were conducted using global Cldn7 knockout mice and inducible, conditional Cldn7 knockout mice., Results: Gene deletion of Cldn7 in intestines showed significant alteration of expression profiles with striking down-regulation of intestinal crypt stem cell markers such as Olfm4, dislocated proliferative cells, and disrupted epithelial cell differentiation. In addition, the isolated Cldn7-deficient crypts where the stem cells reside were either unable to survive at all or formed defective spheroids, highlighting the functional impairment of crypt stem cells in the absence of Cldn7. Remarkably, the Cldn7-expressing organoids with buddings underwent rapid cell degeneration within days after turning off Cldn7 expression in the culture. We identified that activation of Wnt/β-catenin signaling rescued the organoid defects caused by Cldn7 deletion., Conclusions: In this study, we show that Cldn7 is indispensable in controlling Wnt/β-catenin signaling-dependent intestinal epithelial stem cell survival, self-renewal, and cell differentiation. This study could open a door to study roles of TJ proteins in stem cell regulations in other tissues and organs., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Claudin-7 knockout (CLDN7 -/- ) mice display renal salt wasting and dehydration phenotypes. To address the role of CLDN7 in kidneys, we established collecting duct (CD) cell lines from CLDN7 +/+ and CLDN7 -/- mouse kidneys. We found that deletion of CLDN7 increased the transepithelial resistance (TER) and decreased the paracellular permeability for Cl - and Na + in CLDN7 -/- CD cells. Inhibition of transcellular Cl - and Na + channels has no significant effect on TER or dilution potentials. Current-voltage curves were linear in both CLDN7 +/+ and CLDN7 -/- CD cells, indicating that the ion flux was through the paracellular pathway. The impairment of Cl - and Na + permeability phenotype can be rescued by CLDN7 re-expression. We also found that WNK4 (its mutations lead to hypertension) expression, but not WNK1, was significantly increased in CLDN7 -/- CD cell lines as well as in primary CLDN7 -/- CD cells, suggesting that the expression of WNK4 was modulated by CLDN7. In addition, deletion of CLDN7 upregulated the expression level of the apical epithelial sodium channel (ENaC), indicating a potential cross-talk between paracellular and transcellular transport systems. This study demonstrates that CLDN7 plays an important role in salt balance in renal CD cells and modulating WNK4 and ENaC expression levels that are vital in controlling salt-sensitive hypertension.

Background: As a potential tumor suppressor gene, Claudin-7 (Cldn7), which is a component of tight junctions, may play an important role in colorectal cancer occurrence and development., Aims: To generate a knockout mouse model of inducible conditional Cldn7 in the intestine and analyze the phenotype of the mice after induction with tamoxifen., Methods: We constructed Cldn7-flox transgenic mice and crossed them with Villin-CreERT2 mice. The Cldn7 inducible conditional knockout mice appeared normal and were well developed at birth. We induced Cldn7 gene deletion by injecting different dosages of tamoxifen into the mice and then conducted a further phenotypic analysis., Results: After induction for 5 days in succession at a dose of 200 µl tamoxifen in sunflower oil at 10 mg/ml per mouse every time, the mice appeared dehydrated, had a lower temperature, and displayed inactivity or death. The results of hematoxylin-eosin staining showed that the intestines of the Cldn7 inducible conditional knockout mice had severe intestinal defects that included epithelial cell sloughing, necrosis, inflammation and hyperplasia. Owing to the death of ICKO mice, we adjusted the dose of tamoxifen to a dose of 100 µl in sunflower oil at 10 mg/ml per mouse (aged more than 8 weeks old) every 4 days. And we could induce atypical hyperplasia and adenoma in the intestine. Immunofluorescent staining indicated that the intestinal epithelial structure was destroyed. Electron microscopy experimental analysis indicated that the intercellular gap along the basolateral membrane of Cldn7 inducible conditional knockout mice in the intestine was increased and that contact between the cells and matrix was loosened., Conclusions: We generated a model of intestinal Cldn7 inducible conditional knockout mice. Intestinal Cldn7 deletion induced by tamoxifen initiated inflammation and hyperplasia in mice.

Background and Objectives: Tight junction proteins are correlated with cancer development. As the pivotal proteins in epithelial cells, altered expression and distribution of different claudins have been reported in a wide variety of human malignancies. We have previously reported that claudin-7 was strongly expressed in benign bronchial epithelial cells at the cell-cell junction while expression of claudin-7 was either altered with discontinued weak expression or completely absent in lung cancers. Based on these results, we continued working on the expression pattern of claudin-7 and its relationship with lung cancer development. We herein proposed a new Digital Image Classification, Fragmentation index, Morphological analysis (DICFM) method for differentiating the normal lung tissues and lung cancer tissues based on the claudin-7 immunohistochemical staining., Methods: Seventy-seven lung cancer samples were obtained from the Second Affiliated Hospital of Zhejiang University and claudin-7 immunohistochemical staining was performed. Based on C++ and Open Source Computer Vision Library (OpenCV, version 2.4.4), the DICFM processing module was developed. Intensity and fragmentation of claudin-7 expression, as well as the morphological parameters of nuclei were calculated. Evaluation of results was performed using Receiver Operator Characteristic (ROC) analysis., Results: Agreement between these computational results and the results obtained by two pathologists was demonstrated. The intensity of claudin-7 expression was significantly decreased while the fragmentation was significantly increased in the lung cancer tissues compared to the normal lung tissues and the intensity was strongly positively associated with the differentiation of lung cancer cells. Moreover, the perimeters of the nuclei of lung cancer cells were significantly greater than that of the normal lung cells, while the parameters of area and circularity revealed no statistical significance., Conclusions: Taken together, our DICFM approach may be applied as an appropriate approach to quantify the immunohistochemical staining of claudin-7 on the cell membrane and claudin-7 may serve as a marker for identification of lung cancer., (Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: Claudins are a family of tight junction (TJ) membrane proteins involved in a broad spectrum of human diseases including cancer. Claudin-7 is a unique TJ membrane protein in that it has a strong basolateral membrane distribution in epithelial cells and in tissues. Therefore, this study aims to investigate the functional significance of this non-TJ localization of claudin-7 in human lung cancer cells., Methods: Claudin-7 expression was suppressed or deleted by lentivirus shRNA or by targeted-gene deletion. Cell cycle analysis and antibody blocking methods were employed to assay cell proliferation and cell attachment, respectively. Electron microscopy and transepthelial electrical resistance measurement were performed to examine the TJ ultrastructure and barrier function. Co-immunolocalization and co-immunoprecipitation was used to study claudin-7 interaction with integrin β1. Tumor growth in vivo were analyzed using athymic nude mice., Results: Claudin-7 co-localizes and forms a stable complex with integrin β1. Both suppressing claudin-7 expression by lentivirus shRNA in human lung cancer cells (KD cells) and deletion of claudin-7 in mouse lungs lead to the reduction in integrin β1 and phospho-FAK levels. Suppressing claudin-7 expression increases cell growth and cell cycle progression. More significantly, claudin-7 KD cells have severe defects in cell-matrix interactions and adhere poorly to culture plates with a remarkably reduced integrin β1 expression. When cultured on uncoated glass coverslips, claudin-7 KD cells grow on top of each other and form spheroids while the control cells adhere well and grow as a monolayer. Reintroducing claudin-7 reduces cell proliferation, upregulates integrin β1 expression and increases cell-matrix adhesion. Integrin β1 transfection partially rescues the cell attachment defect. When inoculated into nude mice, claudin-7 KD cells produced significantly larger tumors than control cells., Conclusion: In this study, we identified a previously unrecognized function of claudin-7 in regulating cell proliferation and maintaining epithelial cell attachment through engaging integrin β1.

Claudins are a family of tight junction (TJ) integral membrane proteins that play a crucial role in maintaining cell polarity, adhesion, and paracellular permeability. Changes in expression levels of claudin proteins have been associated with human lung cancer. Previously, we have reported that claudin-7 expression is significantly downregulated in human lung carcinomas. To investigate the role of claudin-7 in lung cancer cells after anti-cancer drug treatments, we transfected claudin-7 cDNA into human NCI-H522 lung cancer cells, which have no detectable expression of claudin-7 protein. Flow cytometry analysis demonstrated that cells transfected with claudin-7 had a significantly higher percentage of cell apoptosis when compared to that of vector transfected cell population. The cell viability assayed by MTT and Annexin V was significantly decreased and cell apoptosis was dramatically increased in claudin-7 transfected cells compared to that of vector transfected cells after cisplatin treatment. Cisplatin is an anti-cancer drug clinically used to treat tumors in several tissues including lung tumors. Most importantly, after cisplatin treatment, the expression levels of cleaved caspase-3, -8, and poly adenosine 5'-diphosphate ribose polymerase (PARP) were much higher in claudin-7 transfected cells than in control cells. Furthermore, using the site-directed mutagenesis approach, we identified that claudin-7 was phosphorylated at serine 204 by protein kinase C. Non-phosphorylated claudin-7 mutant showed increased cell viability, suggesting that phosphorylation increases chemosensitivity to cisplatin treatment. We concluded that claudin-7 expression in H522 lung cancer cells increases chemosensitivity to cisplatin through the increased activation of caspase pathway., (© 2013 Japanese Cancer Association.)

Background & Aims: Integrity of the intestinal epithelium is required for nutrition absorption and defense against pathogens. Claudins are cell adhesion molecules that localize at tight junctions (TJs); many are expressed in the intestinal tract, but little is known about their functions. Claudin-7 is unique in that it has a stronger basolateral membrane distribution than other claudins, which localize primarily to apical TJs in the intestinal epithelium. We investigated the basolateral functions of claudin-7 and assessed the effects of disruption of Cldn7 in intestines of mice., Methods: We generated Cldn7(-/-) mice and examined their intestines by histology, molecular and cellular biology, and biochemistry approaches. We performed gene silencing experiments in epithelial cell lines using small interfering RNAs (siRNAs)., Results: The Cldn7(-/-) mice had severe intestinal defects that included mucosal ulcerations, epithelial cell sloughing, and inflammation. Intestines of Cldn7(-/-) mice produced significantly higher levels of cytokines, the nuclear factor κB p65 subunit, and cyclooxygenase 2; they also up-regulated expression of matrix metalloproteinases (MMPs)-3 and -7. siRNA in epithelial cell lines showed that the increased expression of MMP-3 resulted directly from claudin-7 depletion, whereas that of MMP-7 resulted from inflammation. Electron microscopy analysis showed that intestines of Cldn7(-/-) mice had intercellular gaps below TJs and cell matrix loosening. Deletion of Cldn7 reduced expression and altered localization of the integrin α2 subunit in addition to disrupting formation of complexes of claudin-7, integrin α2, and claudin-1 that normally form in epithelial basolateral compartments of intestines., Conclusions: In mice, claudin-7 has non-TJ functions, including maintenance of epithelial cell-matrix interactions and intestinal homeostasis., (Copyright © 2012 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Tight junctions (TJs) are the most apical component of the junctional complexes in mammalian epithelial cells and form selective paracellular barriers restricting the passage of solutes and ions across the epithelial sheets. Claudins, a TJ integral membrane protein family, play a critical role in regulating paracellular barrier permeability. In the in vitro cell culture system, transepithelial electrical resistance (TER) measurement and the flux of radioisotope or fluorescent labeled molecules with different sizes have been widely used to determine the TJ barrier function. In the in vivo system, the tracer molecule Sulfo-NHS-Biotin was initially used in Xenopus embryo system and subsequently was successfully applied to a number of animal tissues in situ and in different organisms under the experimental conditions to examine the functional integrity of TJs by several laboratories. In this chapter, we will describe the detailed procedures of applying biotin as a paracellular tracer molecule to different in vivo systems to assay TJ barrier function.

Epithelial cells are polarized with defined apical tight junctions (TJs), lateral adherens junctions (AJs), and basal integrin–matrix interactions. However, it is increasingly recognized that resident cell junction proteins can be found in varying locations and with previously unrecognized functions. Our study here presents the nanoarchitecture and nanocolocalization of cell junction proteins in culture and tissue by stochastic optical reconstruction microscopy (STORM). The Z‐axial view of noncancerous MDCK‐II and PZ‐HPV‐7 cell–cell junctions resolved β‐catenin and p120ctn localizations to TJs and AJs, with p120ctn apical to β‐catenin and colocalizing with TJ protein claudin‐7. More basally, p120ctn and β‐catenin become colocalized. This topography was lost in isogenic Ras‐transformed MDCK cells and cancerous PC3 cells, where p120ctn becomes basally localized in relation to β‐catenin. Claudin‐7 gene conditional knockout (cKO) in mice also have altered polarity of p120ctn relative to β‐catenin, like that seen in normal‐to‐cancer cell phenotypic transformation. Additionally, claudin‐7 cKO resulted in redistribution and relocalization of other cell junction proteins, including claudin‐1, zonula occludens‐1, integrin α2, epithelial cell adhesion molecule, and focal adhesion kinase (FAK); specifically, integrin α2 and FAK were observed at the apical–lateral compartment. Our data show that STORM reveals regional cellular junction nanoarchitecture previously uncharacterized, providing new insight into potential trans‐compartmental modulation of protein functions. [ABSTRACT FROM AUTHOR]

Background: Necrotizing enterocolitis (NEC) is a leading cause of death in preterm infants. Neonates weighing <1500 grams are at the highest risk for acquiring NEC, with a prevalence of nearly 7-10%, mortality up to 30%, and several long-term complications among survivors. Despite advancements in neonatal medicine, this disease remains a challenge to treat. The aim of this study is to investigate the effect of NEC on gut epithelial tight junctions and its barrier function using a NEC mouse model.Methods: Three-day old C57BL/6 mouse pups were fed with Esbilac formula every 3 hours and then subjected to hypoxia twice daily followed by cold stress. Dam fed pups from the same litters served as controls. Pups were observed and sacrificed 96 hours after the treatments and intestines were removed for experiments. The successful induction of NEC was confirmed by histopathology. Changes in tight junction proteins in NEC intestines were studied by western blotting and immunofluorescent microscopy using specific protein markers. The gut leakage in NEC was visualized using biotin tracer molecules.Results: Our study results demonstrate that we induced NEC in >50% of experimental pups, pups lost nearly 40% of weight and their intestines showed gross changes and microscopic changes associated with NEC. There were inflammatory changes with loss of tight junction barrier function and disruption of tight junction claudin proteins in the intestines of NEC mouse model. We have demonstrated for the first time that NEC intestines develop increased leakiness as visualized by biotin tracer leakage.Conclusions: NEC leads to breakdown of epithelial barrier due to changes in tight junction proteins with increased leakiness which may explain the transmigration of microbes and microbial products from the gut lumen into the blood stream leading to sepsis like signs clinically witnessed. [ABSTRACT FROM AUTHOR]