ABSTRACT
INTRODUCTION: Peritumoral brain edema (PTBE) has been widely reported with many brain tumors, especially with glioma. Since the blood-brain barrier (BBB) is essential for maintaining minimal permeability, any alteration in the interaction of BBB components, specifically in astrocytes and tight junctions (TJ), can result in disrupting the homeostasis of the BBB and making it severely leaky, which subsequently generates edema. OBJECTIVE: This study aimed to evaluate the functional gliovascular unit of the BBB by examining changes in the expression of claudin (CLDN) genes and the expression of transient receptor potential (TRP) membrane channels, additionally to define the correlation between their expressions. The evaluation was conducted using in vitro spheroid swelling models and tumor samples from glioma patients with PTBE. RESULTS: The results of the spheroid model showed that the genes TRPC3, TRPC4, TRPC5, and TRPV1 were upregulated in glioma cells either wild-type isocitrate dehydrogenase 1 (IDH1) or the IDH1 R132H mutant, with or without NaCl treatment. Furthermore, TRP genes appeared to adversely correlate with the up regulation of CLDN1, CLDN3, and CLDN5 genes. Besides, the upregulation of TRPC1 and TRPC4 in IDH1mt-R132H glioma cells. On the other hand, the correlation analysis revealed different correlations between different proteins in PTBE. CLDN1 exhibits a slight positive correlation with CLDN3. Similarly, TRPV1 displays a slight positive correlation with TRPC1. In contrast, TRPC4 shows a slight negative correlation with TRPC5. On the other hand, TRPC3 demonstrates a slight positive correlation with TRPC5, while the non-PTBE analysis highlights a moderate positive correlation between CLDN1 and TRPM4 while CLDN3 exhibits a moderate negative correlation with TRPC4. Additionally, CLDN5 demonstrates a slight negative correlation with TRPC4 but a moderate positive correlation with TRPC3. Furthermore, TRPC1 have a slight negative correlation with TRPV1, TRPC3 exhibiting a slight positive correlation with TRPC4, and TRPV1 showing a slight negative correlation with TRPC5. CONCLUSION: As a conclusion, the current study provided evidence of a slight negative correlation between TRPs and CLDN gene expression in PTBE patients and confirmatory results with some of the genes in cell model of edema.
Subject(s)
Brain Edema , Brain Neoplasms , Claudin-5 , Glioma , Humans , Brain Edema/genetics , Brain Edema/metabolism , Brain Edema/pathology , Glioma/genetics , Glioma/metabolism , Glioma/pathology , Brain Neoplasms/genetics , Brain Neoplasms/metabolism , Brain Neoplasms/pathology , Claudin-5/genetics , Claudin-5/metabolism , Gene Expression Regulation, Neoplastic , Claudin-3/genetics , Claudin-3/metabolism , Blood-Brain Barrier/metabolism , TRPC Cation Channels/genetics , TRPC Cation Channels/metabolism , Isocitrate Dehydrogenase/genetics , Isocitrate Dehydrogenase/metabolism , Cell Line, Tumor , Claudin-1/genetics , Claudin-1/metabolism , Claudins/genetics , Claudins/metabolism , TRPV Cation Channels/genetics , TRPV Cation Channels/metabolism , MaleABSTRACT
Intestinal absorption of phosphate is bimodal, consisting of a transcellular pathway and a poorly characterized paracellular mode, even though the latter one contributes to the bulk of absorption under normal dietary conditions. Claudin-3 (Cldn3), a tight junction protein present along the whole intestine in mice, has been proposed to tighten the paracellular pathway for phosphate. The aim of this work was to characterize the phosphate-related phenotype of Cldn3-deficient mice. Cldn3-deficient mice and wildtype littermates were fed standard diet or challenged for 3 days with high dietary phosphate. Feces, urine, blood, intestinal segments and kidneys were collected. Measurements included fecal, urinary, and plasma concentrations of phosphate and calcium, plasma levels of phosphate-regulating hormones, evaluation of trans- and paracellular phosphate transport across jejunum and ileum, and analysis of intestinal phosphate and calcium permeabilities. Fecal and urinary excretion of phosphate as well as its plasma concentration was similar in both genotypes, under standard and high-phosphate diet. However, Cldn3-deficient mice challenged with high dietary phosphate had a reduced urinary calcium excretion and increased plasma levels of calcitriol. Intact FGF23 concentration was also similar in both groups, regardless of the dietary conditions. We found no differences either in intestinal phosphate transport (trans- or paracellular) and phosphate and calcium permeabilities between genotypes. The intestinal expression of claudin-7 remained unaltered in Cldn3-deficient mice. Our data do not provide evidence for a decisive role of Cldn3 for intestinal phosphate absorption and phosphate homeostasis. In addition, our data suggest a novel role of Cldn3 in regulating calcitriol levels.
Subject(s)
Claudin-3 , Fibroblast Growth Factor-23 , Intestinal Absorption , Phosphates , Animals , Phosphates/metabolism , Phosphates/urine , Mice , Claudin-3/metabolism , Claudin-3/genetics , Fibroblast Growth Factor-23/metabolism , Calcitriol/metabolism , Calcitriol/blood , Calcium/metabolism , Mice, Inbred C57BL , Male , Mice, Knockout , Fibroblast Growth Factors/metabolism , Fibroblast Growth Factors/genetics , Intestinal Mucosa/metabolismABSTRACT
OBJECTIVES: As a critical component of the epithelial barrier, tight junctions (TJs) are essential in nasal mucosa against pathogen invasion. However, the function of TJs has rarely been reported in nasal inverted papilloma (NIP). This study aims to investigate the potential factors of TJs' abnormality in NIP. METHODS: We assessed the expression of ZO-1, occludin, claudin-1, claudin-3, and claudin-7 in healthy controls and NIP by real-time quantitative polymerase chain reaction and immunofluorescent staining. The correlation between TJs expression and neutrophil count, TH 1/TH 2/TH 17 and regulatory T cell biomarkers, and the proportion of nasal epithelial cells was investigated. RESULTS: Upregulation of ZO-1, occludin, claudin-1, and claudin-7, along with downregulation of claudin-3, was found in NIP compared to control (all p < 0.05). An abnormal proportion with a lower number of ciliated cells (control vs. NIP: 37.60 vs. 8.67) and goblet cells (12.52 vs. 0.33) together with a higher number of basal cells (45.58 vs. 124.00) in NIP. Meanwhile, claudin-3 was positively correlated with ciliated and goblet cells (all p < 0.01). Additionally, neutrophils were excessively infiltrated in NIP, negatively correlated with ZO-1, but positively with claudin-3 (all p < 0.05). Furthermore, FOXP3, IL-10, TGF-ß1, IL-5, IL-13, and IL-22 levels were induced in NIP (all p < 0.01). Occludin level was negatively correlated with IL-10, IL-5, IL-13, and IL-22, whereas ZO-1 was positively with TGF-ß1 (all p < 0.05). CONCLUSION: Nasal epithelial barrier dysfunction with TJs anomalies is commonly associated with abnormal proliferation and differentiation of epithelial cells and imbalance of immune and inflammatory patterns in NIP. LEVEL OF EVIDENCE: NA Laryngoscope, 134:552-561, 2024.
Subject(s)
Papilloma, Inverted , Tight Junctions , Humans , Interleukin-10/metabolism , Transforming Growth Factor beta1/metabolism , Occludin/metabolism , Interleukin-13/metabolism , Claudin-1/metabolism , Claudin-3/genetics , Claudin-3/metabolism , Interleukin-5/metabolism , Epithelial Cells/metabolismABSTRACT
Dysregulation of both the gut barrier and microbiota (dysbiosis) promotes susceptibility to and severity of Inflammatory Bowel Diseases (IBD). Leaky gut and dysbiosis often coexist; however, potential interdependence and molecular regulation are not well understood. Robust expression of claudin-3 (CLDN3) characterizes the gut epithelium, and studies have demonstrated a positive association between CLDN3 expression and gut barrier maturity and integrity, including in response to probiotics. However, the exact status and causal role of CLDN3 in IBD and regulation of gut dysbiosis remain unknown. Analysis of mouse and human IBD cohorts helped examine CLDN3 expression in IBD. The causal role was determined by modeling CLDN3 loss of expression during experimental colitis. 16S sequencing and in silico analysis helped examine gut microbiota diversity between Cldn3KO and WT mice and potential host metabolic responses. Fecal microbiota transplant (FMT) studies were performed to assess the role of gut dysbiosis in the increased susceptibility of Cldn3KO mice to colitis. A significant decrease in CLDN3 expression characterized IBD and CLDN3 loss of expression promoted colitis. 16S sequencing analysis suggested gut microbiota changes in Cldn3KO mice that were capable of modulating fatty acid metabolism and oxidative stress response. FMT from naïve Cldn3KO mice promoted colitis susceptibility in recipient germ-free mice (GFM) compared with GFM-receiving microbiota from WT mice. Our data demonstrate a critical role of CLDN3 in maintaining normal gut microbiota and inflammatory responses, which can be harnessed to develop novel therapeutic opportunities for patients with IBD.
Subject(s)
Claudin-3 , Colitis , Gastrointestinal Microbiome , Inflammatory Bowel Diseases , Humans , Claudin-3/genetics , Colitis/genetics , Colitis/complications , Dysbiosis/complications , Fecal Microbiota Transplantation , Inflammatory Bowel Diseases/complications , Animals , MiceABSTRACT
BACKGROUND: Claudins (CLDNs), major components of tight junctions, control paracellular permeabilities of mineral ions and wastes. The absorption of nutrients including glucose and amino acids (AAs) is regulated by intestinal epithelial cells. However, the role of CLDNs is not fully understood. OBJECTIVES: The purpose of this study was to clarify the effect of AA deprivation on the expression of AA transporters and CLDNs, as well as the role of CLDNs in the regulation of paracellular AA fluxes. METHODS: The messenger RNA and protein expression of various CLDNs were examined by real-time quantitative polymerase chain reaction and Western blot analyses, respectively. The AA selectivity of CLDNs was estimated using liquid chromatography-tandem mass spectrometry (LC-MS) analysis. RESULTS: The expression levels of some AA transporters, CLDN4, and CLDN15 were increased by AA deprivation in normal mouse colon-derived MCE301 cells. The expression of AA transporters and CLDN15 in the mouse colon was positively correlated with aging but the expression of CLDN4 was not. The AA deprivation-induced elevation of CLDN4 expression was inhibited by MHY1485, a mammalian target of rapamycin (mTOR) activator. Furthermore, CLDN4 expression was increased by rapamycin, an mTOR inhibitor. mTOR may be involved in the transcriptional activation of CLDN4. The fluxes of AAs from the basal to apical compartments were decreased and increased by CLDN4 overexpression and silencing, respectively. LC-MS analysis showed that the fluxes of all AAs, especially Lys, His, and Arg, were enhanced by CLDN4 silencing. CONCLUSIONS: CLDN4 is suggested to form a paracellular barrier to AAs, especially alkaline AAs, which is attenuated with aging.
Subject(s)
Amino Acids , Claudins , Animals , Mice , Amino Acids/metabolism , Claudin-3/genetics , Claudin-3/metabolism , Claudin-4/genetics , Claudin-4/metabolism , Claudins/genetics , Claudins/metabolism , Mammals/metabolism , Tight Junctions , TOR Serine-Threonine Kinases/metabolismABSTRACT
Radiation therapy and unwanted radiological or nuclear exposure, such as nuclear plant accidents, terrorist attacks, and military conflicts, pose serious health issues to humans. Dysfunction of the intestinal epithelial barrier and the leakage of luminal antigens and bacteria across the barrier have been linked to various human diseases. Intestinal permeability is regulated by intercellular structures, termed tight junctions (TJs), which are disrupted after radiation exposure. In this study, we investigated radiation-induced alterations in TJ-related proteins in an intestinal epithelial cell model. Caco-2 cells were irradiated with 2, 5, and 10 Gy and harvested 1 and 24 h after X-ray exposure. The trypan blue assay revealed that cell viability was reduced in a dose-dependent manner 24 h after X-ray exposure compared to that of non-irradiated cells. However, the WST-8 assay revealed that cell proliferation was significantly reduced only 24 h after radiation exposure to 10 Gy compared to that of non-irradiated cells. In addition, a decreased growth rate and increased doubling time were observed in cells irradiated with X-rays. Intestinal permeability was significantly increased, and transepithelial electrical resistance values were remarkably reduced in Caco-2 cell monolayers irradiated with X-rays compared to non-irradiated cells. X-ray irradiation significantly decreased the mRNA and protein levels of ZO-1, occludin, claudin-3, and claudin-4, with ZO-1 and claudin-3 protein levels decreasing in a dose-dependent manner. Overall, the present study reveals that exposure to X-ray induces dysfunction of the human epithelial intestinal barrier and integrity via the downregulation of TJ-related genes, which may be a key factor contributing to intestinal barrier damage and increased intestinal permeability.
Subject(s)
Intestinal Diseases , Intestinal Mucosa , Humans , Caco-2 Cells , Intestinal Mucosa/metabolism , X-Rays , Claudin-3/genetics , Claudin-3/metabolism , Intestines , Epithelial Cells/metabolism , Intestinal Diseases/metabolism , PermeabilityABSTRACT
Glioblastoma multiforme (GBM) is a significantly malignant and lethal brain tumor with an average survival time of less than 12 months. Several researches had shown that Claudin-3 (CLDN3) is overexpressed in various cancers and might be important in their growth and spread. In this study, we used qRT-PCR, western blotting, immunohistochemistry, and immunofluorescence staining assays to investigate the expression levels of various proteins. To explore the proliferation abilities of GBM cells, we conducted the CCK-8 and EdU-DNA formation assays. Wound healing and transwell assays were used to investigate the capacities of invasion and migration of GBM cells. Additionally, we constructed an intracranial xenograft model of GBM to study the in vivo role of CLDN3. Our study devoted to investigate the function of CLDN3 in the pathogenesis and progression of GBM. Our study revealed that CLDN3 was upregulated in GBM and could stimulate tumor cell growth and epithelial-mesenchymal transition (EMT) in both laboratory and animal models. We also discovered that CLDN3 expression could be triggered by transforming growth factor-ß (TGF-ß) and reduced by specific inhibitors of the TGF-ß signaling pathway, such as ITD-1. Further analysis revealed that increased CLDN3 levels enhanced TGF-ß-induced growth and EMT in GBM cells, while reducing CLDN3 levels weakened these effects. Our study demonstrated the function of CLDN3 in facilitating GBM growth and metastasis and indicated its involvement in the tumorigenic effects of TGF-ß. Developing specific inhibitors of CLDN3 might, therefore, represent a promising new approach for treating this devastating disease.
Subject(s)
Brain Neoplasms , Claudin-3 , Glioblastoma , Animals , Humans , Brain Neoplasms/pathology , Cell Line, Tumor , Cell Movement , Cell Proliferation , Claudin-3/genetics , Claudin-3/metabolism , Epithelial-Mesenchymal Transition , Glioblastoma/genetics , Transforming Growth Factor betaABSTRACT
Hair follicles (HFs) undergo cyclic phases of growth, regression, and rest in association with hair shafts to maintain the hair coat. Nonsense mutations in the tight junction protein claudin (CLDN)-1 cause hair loss in humans. Therefore, we evaluated the roles of CLDNs in hair retention. Among the 27 CLDN family members, CLDN1, CLDN3, CLDN4, CLDN6, and CLDN7 were expressed in the inner bulge layer, isthmus, and sebaceous gland of murine HFs. Hair phenotypes were observed in Cldn1 weaker knockdown and Cldn3-knockout (Cldn1Δ/Δ Cldn3-/- ) mice. Although hair growth was normal, Cldn1Δ/Δ Cldn3-/- mice showed striking hair loss in the first telogen. Simultaneous deficiencies in CLDN1 and CLDN3 caused abnormalities in telogen HFs, such as an aberrantly layered architecture of epithelial cell sheets in bulges with multiple cell layers, mislocalization of bulges adjacent to sebaceous glands, and dilated hair canals. Along with the telogen HF abnormalities, which shortened the hair retention period, there was an enhanced proliferation of the epithelium surrounding HFs in Cldn1Δ/Δ Cldn3-/- mice, causing accelerated hair regrowth in adults. Our findings suggested that CLDN1 and CLDN3 may regulate hair retention in infant mice by maintaining the appropriate layered architecture of HFs, a deficiency of which can lead to alopecia.
Subject(s)
Alopecia , Animals , Mice , Alopecia/genetics , Claudin-1/genetics , Claudin-1/metabolism , Claudin-3/genetics , Claudin-3/metabolism , Claudin-4/metabolism , Mutation , AgingABSTRACT
Claudin-4 (CLDN4) is a key component of tight junctions (TJs) in epithelial cells. CLDN4 is overexpressed in many epithelial malignancies and correlates with cancer progression. Changes in CLDN4 expression have been associated with epigenetic factors (such as hypomethylation of promoter DNA), inflammation associated with infection and cytokines, and growth factor signaling. CLDN4 helps to maintain the tumor microenvironment by forming TJs and acts as a barrier to the entry of anticancer drugs into tumors. Decreased expression of CLDN4 is a potential marker of epithelial-mesenchymal transition (EMT), and decreased epithelial differentiation due to reduced CLDN4 activity is involved in EMT induction. Non-TJ CLDN4 also activates integrin beta 1 and YAP to promote proliferation, EMT, and stemness. These roles in cancer have led to investigations of molecular therapies targeting CLDN4 using anti-CLDN4 extracellular domain antibodies, gene knockdown, clostridium perfringens enterotoxin (CPE), and C-terminus domain of CPE (C-CPE), which have demonstrated the experimental efficacy of this approach. CLDN4 is strongly involved in promoting malignant phenotypes in many epithelial cancers and is regarded as a promising molecular therapeutic target.
Subject(s)
Antineoplastic Agents , Neoplasms , Claudin-4/genetics , Claudin-4/metabolism , Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic use , Antineoplastic Agents/metabolism , Tight Junctions/metabolism , Epithelial Cells/metabolism , Signal Transduction , Claudin-3/genetics , Enterotoxins/pharmacology , Cell Line, Tumor , Neoplasms/drug therapy , Neoplasms/genetics , Neoplasms/metabolismABSTRACT
Castration-resistant prostate cancer (CRPC) development is the foremost concern after treatment of patients with high risk with locally advanced or metastatic prostate cancer. Androgen receptor (AR) is the main driver of CRPC development, through its interaction with epigenetic modifier genes, placing epigenetics modifications in the forefront of CRPC development. Comparing the DNA methylation and expression profile of androgen-sensitive and -refractory prostate cancer cells, we describe the epigenetic silencing of claudin-3 (CLDN3) in AR positive cells resistant to androgen deprivation (LNCaP-abl). CLDN3 silencing was associated with DNA methylation, loss of histone acetylation and H3K27 methylation, and was re-expressed by the combined treatment with the epigenetic modulators Aza and SAHA. From a functional point of view, CLDN3 loss was associated with increased cellular invasion. Immunohistochemical analysis showed decreased CLDN3 expression in samples from CRPC patients. Interestingly, CLDN3 expression was significantly decreased in samples from patients with high total Gleason score (≥8) and locally advanced tumors. Finally, CLDN3 loss of expression was associated with worse disease-free survival and time to clinical progression. In conclusion, our findings strongly indicate that epigenetic silencing of CLDN3 is a common event in CRPC that could be useful as a molecular marker for the prognosis of prostate cancer patients and to discriminate aggressive from indolent prostate tumors.
Subject(s)
Prostatic Neoplasms, Castration-Resistant , Male , Humans , Prostatic Neoplasms, Castration-Resistant/drug therapy , Claudin-3/genetics , Androgen Antagonists/therapeutic use , Androgens/therapeutic use , Prognosis , Receptors, Androgen/metabolism , Cell Line, TumorABSTRACT
Claudin-3 is a tight junction protein that has often been associated with the progression and metastasis of various tumors. Here, the role of claudin-3 in tumor-induced lymphangiogenesis is investigated. We found an increased lymphangiogenesis in the B16F10 tumor in claudin-3 knockout mice, accompanied by augmented melanoma cell metastasis into sentinel lymph nodes. In vitro, the overexpression of claudin-3 on lymphatic endothelial cells inhibited tube formation by suppressing cell migration, resulting in restricted lymphangiogenesis. Further experiments showed that claudin-3 inhibited lymphatic endothelial cell migration by regulating the PI3K signaling pathway. Interestingly, the expression of claudin-3 in lymphatic endothelial cells is down-regulated by vascular endothelial growth factor C that is often present in the tumor microenvironment. This study indicates that claudin-3 plays an important role as a signaling molecule in lymphatic endothelial cell activity associated with tumor lymphangiogenesis, which may further contribute to melanoma metastasis.
Subject(s)
Claudin-3 , Lymphatic Vessels , Melanoma , Animals , Mice , Claudin-3/genetics , Claudin-3/metabolism , Endothelial Cells/metabolism , Lymphangiogenesis , Lymphatic Metastasis/pathology , Lymphatic Vessels/metabolism , Melanoma/pathology , Phosphatidylinositol 3-Kinases/metabolism , Signal Transduction , Tumor Microenvironment , Vascular Endothelial Growth Factor C/metabolismABSTRACT
PURPOSE: This study aims to explore the pivotal mediating role of adenosine monophosphate-activated protein kinase (AMPK) in liver tight junctions and liver regeneration of a partial hepatectomy (PH) mouse model. METHODS: A 70% PH mouse model was used. Firstly, mice were randomly divided into sham, 70% PH, AMPK-activated, and AMPK-inhibited groups. Then serum levels of alanine aminotransferase, aspartate transaminase, total bilirubin, direct bilirubin, albumin, and prealbumin were tested on postoperative days 1, 2 and 3. Furthermore, the expression of tight junction proteins like occludin, claudin-3, and ZO-1, together with bile salt export pump (BSEP), which reflects liver function, and AMPK were measured by Western blot and quantitative real-time polymerase chain reaction. Moreover, the expression of tight junction proteins, BSEP, and Ki-67 were examined by immunohistochemistry. RESULTS: After 70% PH, without intervention, the changes in expression of hepatic tight junction proteins (occludin, claudin-3, and ZO-1) were consistent with that of BSEP, which could reflect liver function. After treatment with AMPK activator, the high expression status of tight junction proteins occurred in advance and was maintained stably and for a longer time. It was beneficial to liver function and liver regeneration was promoted at early periods and enhanced continuously after PH. CONCLUSIONS: Activation of AMPK could effectively enhance the expression of hepatic tight junction proteins after PH. Therefore, it could speed up the recovery of liver function and promote liver regeneration especially early after PH.
Subject(s)
Focal Nodular Hyperplasia , Liver Regeneration , Mice , Animals , Liver Regeneration/physiology , Hepatectomy , Tight Junctions/metabolism , Occludin/metabolism , Occludin/pharmacology , AMP-Activated Protein Kinases/metabolism , AMP-Activated Protein Kinases/pharmacology , Claudin-3/genetics , Claudin-3/metabolism , Adenosine Monophosphate , Zonula Occludens-1 Protein/metabolism , Tight Junction Proteins/metabolism , Disease Models, Animal , Bilirubin/metabolismABSTRACT
A change in claudin expression has been demonstrated in various tumors. The present study specifically compares claudin expression in oral squamous cell carcinoma (OSCC) with healthy oral epithelium from the same individual and analyzes the association between claudin expression and the clinically relevant course parameters. Our study includes tissue samples and clinically relevant follow-up data from 60 patients with primary and untreated OSCC. The oral mucosa was analyzed via Western blot for the expression of claudin-1, -2, -3, -4, -5, and -7. Importantly, the tumor and healthy tissues were obtained pairwise from patients, allowing for intraindividual comparisons. Both the healthy and tumor epithelium from the oral cavity did not express the claudin-3 protein. The intraindividual comparison revealed that, in OSCC, claudin-2 expression was higher, and the expression of claudin-4, -5, and -7 was lower than in healthy epithelium. An association was found between increased claudin-2 expression and shorter relapse-free survival. In addition, the reduced expression of claudin-4 had a negative impact on relapse-free survival. Furthermore, associations between the reduced expression of claudin-7 and the stage of a tumor, or the presence of lymph node metastases, were found. Thus, the expression level of claudin-2, -4, and -7 appears to be predictive of the diagnosis and prognosis of OSCC.
Subject(s)
Carcinoma, Squamous Cell , Head and Neck Neoplasms , Mouth Neoplasms , Biomarkers, Tumor/metabolism , Carcinoma, Squamous Cell/metabolism , Claudin-1/metabolism , Claudin-2 , Claudin-3/genetics , Claudin-4/genetics , Claudins/genetics , Claudins/metabolism , Humans , Immunohistochemistry , Mouth Neoplasms/metabolism , Neoplasm Recurrence, Local , Squamous Cell Carcinoma of Head and NeckABSTRACT
Claudin-based tight junctions (TJs) are formed at the most apical part of cell-cell contacts in epithelial cells. Previous studies suggest that scaffolding proteins ZO-1 and ZO-2 (ZO proteins) determine the location of TJs by interacting with claudins, but this idea is not conclusive. To address the role of the ZO proteins binding to claudins at TJs, a COOH-terminal PDZ domain binding motif-deleted claudin-3 mutant, which lacks the ZO protein binding, was stably expressed in claudin-deficient MDCK cells. The COOH-terminus-deleted claudin-3 was localized at the apicolateral region similar to full-length claudin-3. Consistently, freeze-fracture electron microscopy revealed that the COOH-terminus-deleted claudin-3-expressing cells reconstituted belts of TJs at the most apical region of the lateral membrane and restored functional epithelial barriers. These results suggest that the interaction of claudins with ZO proteins is not a prerequisite for TJ formation at the most apical part of cell-cell contacts.
Subject(s)
Claudins , Tight Junctions , Cell Line , Claudin-1/metabolism , Claudin-3/genetics , Claudin-3/metabolism , Claudin-5/metabolism , Claudins/genetics , Claudins/metabolism , Humans , PDZ Domains , Protein Binding , Tight Junctions/metabolismABSTRACT
Strains of Clostridium perfringens produce a two-domain enterotoxin (CpE) that afflicts humans and domesticated animals, causing prevalent gastrointestinal illnesses. CpE's C-terminal domain (cCpE) binds cell surface receptors, followed by a restructuring of its N-terminal domain to form a membrane-penetrating ß-barrel pore, which is toxic to epithelial cells of the gut. The claudin family of membrane proteins are known receptors for CpE and also control the architecture and function of cell-cell contacts (tight junctions) that create barriers to intercellular molecular transport. CpE binding and assembly disables claudin barrier function and induces cytotoxicity via ß-pore formation, disrupting gut homeostasis; however, a structural basis of this process and strategies to inhibit the claudin-CpE interactions that trigger it are both lacking. Here, we used a synthetic antigen-binding fragment (sFab) library to discover two sFabs that bind claudin-4 and cCpE complexes. We established these sFabs' mode of molecular recognition and binding properties and determined structures of each sFab bound to claudin-4-cCpE complexes using cryo-EM. The structures reveal that the sFabs bind a shared epitope, but conform distinctly, which explains their unique binding equilibria. Mutagenesis of antigen/sFab interfaces observed therein result in binding changes, validating the structures, and uncovering the sFab's targeting mechanism. From these insights, we generated a model for CpE's claudin-bound ß-pore that predicted sFabs would not prevent cytotoxicity, which we then verified in vivo. Taken together, this work demonstrates the development and mechanism of claudin/cCpE-binding sFabs that provide a framework and strategy for obstructing claudin/CpE assembly to treat CpE-linked gastrointestinal diseases.
Subject(s)
Claudins , Enterotoxins , Animals , Claudin-3/genetics , Claudin-3/metabolism , Claudin-4/genetics , Claudin-4/metabolism , Claudins/metabolism , Clostridium perfringens , Enterotoxins/metabolism , Epitopes/metabolism , Humans , Protein BindingABSTRACT
The composition of cell contacts in the endometrium plays an important role in the process of embryo implantation and the establishment of pregnancy. In previous studies, we showed an induction of the tight junction protein claudin-3 in the developing decidua from day 6.5 of pregnancy onward. To evaluate the role of this specific claudin-3 distribution, we here evaluated the effect of an endometrial claudin-3 deletion in implantation and embryo development in claudin-3 knockout mice. Claudin-3 knockout mice were fertile but revealed a slightly reduced amount of implantation sites as well as of litter size. Though implantation sites showed morphologically regularly developed embryos and deciduas, depth of ectoplacental cone invasion was reduced in tendency compared to controls. The weight of the implantation sites on day 6.5 and 8.5 of pregnancy as well as the weight of the embryos on day 17.5 of pregnancy, but not of the placentas, was significantly reduced in claudin-3 knockout mice due to a maternal effect. This could be due to an impairment of decidualization as substantiated by a downregulation of the transcription of various decidua-associated genes in the early implantation sites of claudin-3 knockout mice. The fact that claudin-3 knockout mice are nevertheless fertile possibly may be compensated by the presence of other claudins like claudin-4 and claudin-10.
Subject(s)
Decidua , Embryo Implantation , Animals , Claudin-3/genetics , Claudin-3/metabolism , Claudin-4/metabolism , Claudins/genetics , Claudins/metabolism , Decidua/metabolism , Embryo Implantation/genetics , Endometrium/metabolism , Female , Mice , Mice, Knockout , Pregnancy , Stromal Cells/metabolismABSTRACT
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.
Subject(s)
Ovarian Neoplasms , Carcinoma, Ovarian Epithelial/therapy , Cell Line, Tumor , Claudin-3/genetics , Claudin-3/metabolism , Claudin-4/genetics , Claudin-4/metabolism , Claudins/genetics , Claudins/metabolism , Claudins/therapeutic use , Female , Humans , Ovarian Neoplasms/diagnosis , Ovarian Neoplasms/genetics , Ovarian Neoplasms/therapyABSTRACT
Claudins are a family of tight junction proteins expressed in epithelial tissues during development and in postnatal life. We hypothesized that claudins are required for branching morphogenesis in the developing chick lung. To test this hypothesis, we exposed cultured chick lung explants at embryonic day 5 to a truncated non-toxic form of the Clostridium perfringens enterotoxin known as C-CPE that removes C-CPE-sensitive claudins from tight junctions. Using in situ hybridization and immunofluorescence studies, we established that only one C-CPE-sensitive claudin, Claudin-3, was expressed in the chick lung at this stage. C-CPE treated lung explants did not exhibit any defect in lung branching compared to controls. However, they did exhibit a significantly smaller lumen area, suggesting that paracellular permeability was perturbed. The decrease in lumen area was associated with a loss of Claudin-3 expression within tight junctions of the respiratory epithelium and an increase in permeability of the respiratory epithelium. When C-CPE-treated lung explants were treated with forskolin, lumen area was restored. In summary, removal of a sealing claudin, Claudin-3, from tight junctions in embryonic lung epithelium results in a decrease in lumen area and in hydrostatic pressure needed for lung development.
Subject(s)
Chickens , Claudins , Animals , Claudin-3/genetics , Claudins/genetics , Epithelium , LungABSTRACT
OBJECTIVES: Network of long noncoding RNA-microRNA (miRNA)-mRNA is becoming increasingly pivotal roles in carcinogenesis mechanism. Herein, we aim to delineate the mechanistic understanding of dipeptidyl peptidase like 10-antisense RNA 1 (DPP10-AS1)/miRNA-324-3p/claudin 3 (CLDN3) axis in the malignancy of pancreatic cancer (PC). METHODS: Microarray profiling and other bioinformatics methods were adopted to predict differentially expressed long noncoding RNA-miRNA-mRNA in PC, followed by verification of expression of DPP10-AS1, microRNA-324-3p (miR-324-3p), and CLDN3 in PC cells. The relationship among DPP10-AS1, miR-324-3p, and CLDN3 were further assessed. The PC cell invasion and migration were evaluated by scratch test and transwell assay. Tumor formation and lymph node metastasis were assessed in nude mice. RESULTS: Highly expressed DPP10-AS1 and CLDN3 and poorly expressed miR-324-3p were identified in PC cells. The competitively binding between DPP10-AS1 and miR-324-3p was identified, and CLDN3 was targeted and downregulated by miR-324-3p. In addition, DPP10-AS1 was found to sequester miR-324-3p to release CLDN3 expression. DPP10-AS1 knockdown or miR-324-3p restoration diminished migration, invasion, tumor formation, microvessel density, and lymph node metastasis of PC cells, which was associated with CLDN3 downregulation. CONCLUSIONS: Taken together, the study identified the regulatory role of DPP10-AS1/miR-324-3p/CLDN3 axis in PC, offering a mechanistic basis suggesting DPP10-AS1 ablation as a therapeutic target against PC.
Subject(s)
MicroRNAs , Pancreatic Neoplasms , RNA, Long Noncoding , Animals , Mice , Claudin-3/genetics , Claudin-3/metabolism , Down-Regulation , RNA, Long Noncoding/genetics , Lymphatic Metastasis , Mice, Nude , Cell Line, Tumor , MicroRNAs/genetics , MicroRNAs/metabolism , Pancreatic Neoplasms/genetics , Pancreatic Neoplasms/pathology , Cell Proliferation/genetics , Gene Expression Regulation, Neoplastic , Cell Movement/genetics , Pancreatic NeoplasmsABSTRACT
In lactating mammary glands, alveolar mammary epithelial cells (MECs) produce milk and form less-permeable tight junctions (TJs). However, alveolar TJs are weakened with a reduction in milk production in mammary glands due to mastitis or weaning in the presence of high levels of IL-1ß, IL-6, or TNF-α. In this study, using in vitro cultured model of MECs with milk-producing ability and lactation-specific TJs, we investigated whether the aforementioned cytokines affect MEC TJs. The results showed that TNF-α, IL-1ß, and IL-6 affected lactation-specific TJs in different ways. In particular, upon activation of p38 and JNK signalling, IL-1ß caused rapid disruption of TJs at tricellular contact points. IL-1ß treatment led to decreased CLDN3, CLDN4, and OCLN levels and a weakened TJ barrier. The adverse effects of IL-1ß on TJs were mimicked by anisomycin, which is an activator of p38 and JNK signalling, and were blocked by MEC pretreatment with a p38 inhibitor but not a JNK inhibitor. The mislocalization of tricellulin at tricellular contact areas was confirmed in MECs treated with IL-1ß or anisomycin. These results indicate that IL-1ß is a key cytokine that adversely affects the TJs between MECs by activating p38.