Changes In Expression Of Claudins Research Articles

Intestinal Barrier, Claudins and Mycotoxins.

The intestinal barrier is the main barrier against all of the substances that enter the body. Proper functioning of this barrier guarantees maintained balance in the organism. Mycotoxins are toxic, secondary fungi metabolites, that have a negative impact both on human and animal health. It was postulated that various mycotoxins may affect homeostasis by disturbing the intestinal barrier. Claudins are proteins that are involved in creating tight junctions between epithelial cells. A growing body of evidence underlines their role in molecular response to mycotoxin-induced cytotoxicity. This review summarizes the information connected with claudins, their association with an intestinal barrier, physiological conditions in general, and with gastrointestinal cancers. Moreover, this review also includes information about the changes in claudin expression upon exposition to various mycotoxins.

Developmental Changes in Mechanisms Mediating Phosphate Homeostasis

Background Phosphate (Pi) is a multivalent ion involved in a multitude of physiological processes including the formation of hydroxyapatite, the core structural component of bone. Hydroxyapatite formation occurs rapidly early in mammalian development enabling a high rate of bone formation. In the small intestine Pi absorption can occur by a transcellular or passive paracellular pathway. Of note, paracellular ion flux is governed largely by a family of proteins known as claudins, which confer permeability to epithelia. In the kidney, Pi reabsorption occurs entirely through the transcellular route. Pathological perturbations in Pi absorption and reabsorption can result in impaired bone formation and mineralization. We therefore aimed to characterize the normal developmental progression of Pi handling in the gut and kidney to determine mechanisms by which the developing mammal establishes a positive Pi balance. Methods Gene expression was determined by real-time PCR of wild-type mice aged 1, 7 days, 14 and 28 days as well as 2, 3 and 6 months of age. Of note, mice were weaned at 22 days of age. To assess developmental changes in paracellular Pi permeability, small bowel segments were isolated from mice aged 9-14 days old or 15-17 weeks of age and Chloride-Pi diffusion potentials assessed in Ussing chambers. Results The secondary sodium-Pi transporters NaPiIIb in the intestine and NaPiIIa, NaPiIIc in the kidney all showed peak expression levels early in development and proceeded to decrease in their expression levels into adulthood. However, unlike NaPiIIb which consistently displayed high expression before weaning age, NaPiIIa and NaPiIIc expression showed a lifetime low during early suckling followed by maximum lifetime expression at 2 weeks of age. There were no specific changes in claudin expression throughout development. Consistent with this, paracellular Pi permeability did not change between 9-14 days and 15-17 months of age along the intestine. Discussion Our results suggest that the mechanism responsible for establishing a positive Pi balance in young mammals is via increased transcellular absorption/reabsorption, whereas paracellular Pi flux is unlikely a significant contributor.

Abstract 4036: Differential expression of claudins between benign and malignant salivary gland tumors

Abstract Salivary gland tumors comprise a heterogeneous group of lesions presenting different histological features and diverse clinical behavior. Pleomorphic adenoma is the most common benign tumor of major salivary glands. It shows a marked histological diversity with epithelial, myoepithelial, and mesenchymal components in a variety of patterns. Although it presents a slow progression, without treatment can cause significant morbidity and even death. Mucoepidermoid carcinoma is the most common malignant tumor of the salivary glands. Local recurrence rates range from 7% to 26% and distant metastasis rates range from 6% to 15%. Tight junctions (TJ) are considered one of the major structures in the establishment and maintenance of cell polarity having important physiological function in the formation of tubular organs, including the salivary glands. Some studies have demonstrated an association between tumor behavior and protein expression of tight junctions, particularly claudins. Claudins are transmembrane proteins important for the maintenance of cell adhesion and polarity and act as selective barriers. Changes in claudin expression have been detected in several tumors and appear to be related to progression and invasion. However, few studies have evaluated the role of claudins in the development of salivary gland tumors. In this study, using Real Time RT-PCR we have analyzed the expression of claudins-1, -3, -4, -5, -7, -11 in a series of 40 salivary gland tumor samples (20 cases of pleomorphic adenoma and 20 cases of mucoepidermoid carcinoma) and 10 samples of normal salivary glands tissue samples. Increased expression of claudin-1 and claudin-7 was observed in mucoepidermoid carcinoma when compared to pleomorphic adenoma (p=0.010 and p=0.001, respectively). Decreased expression of claudin-3 and claudin-11 was observed in mucoepidermoid carcinoma when compared to pleomorphic adenoma (p=0.005 and p=0.030, respectively). Regarding mucoepidermoid carcinoma tissue samples, we have evaluated the presence/absence of CRTC1/MAML2 fusion and demonstrated that 57.9% of the cases were positive for the presence of the fusion. Claudin-7 expression was associated with the presence of the fusion (p=0.048) and the absence of the fusion was associated with high histological grade tumors (p=0.023). Our study provides evidence of alterations in the expression of claudins in salivary gland tumors demonstrating that regulation of tight junction components is an important feature in salivary gland tumors behavior. CNPq Research Grant 443243/2014-0 Citation Format: Maria L. Arrojo, Claudia Arruda, Luiz P. Kowalski, Fernando A. Soares, Silvia V. Lourenço, Cláudia M. Coutinho-Camillo. Differential expression of claudins between benign and malignant salivary gland tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4036.

The claudin family of proteins in human malignancy: a clinical perspective

Tight junctions, or zonula occludens, are the most apical component of the junctional complex and provide one form of cell–cell adhesion in epithelial and endothelial cells. Nearly 90% of malignant tumors are derived from the epithelium. Loss of cell–cell adhesion is one of the steps in the progression of cancer to metastasis. At least three main tight junction family proteins have been discovered: occludin, claudin, and junctional adhesion molecule (JAM). Claudins are the most important structural and functional components of tight junction integral membrane proteins, with at least 24 members in mammals. They are crucial for the paracellular flux of ions and small molecules. Overexpression or downregulation of claudins is frequently observed in epithelial-derived cancers. However, molecular mechanisms by which claudins affect tumorigenesis remain largely unknown. As the pivotal proteins in epithelial cells, altered expression and distribution of different claudins have been reported in a wide variety of human malignancies, including pancreatic, colonic, lung, ovarian, thyroid, prostate, esophageal, and breast cancers. In this review, we will give the readers an overall picture of the changes in claudin expression observed in various cancers and their mechanisms of regulation. Downregulation of claudins contributes to epithelial transformation by increasing the paracellular permeability of nutrients and growth factors to cancerous cells. In the cases of upregulation of claudin expression, the barrier function of the cancerous epithelia changes, as they often display a disorganized arrangement of tight junction strands with increased permeability to paracellular markers. Finally, we will summarize the literature suggesting that claudins may become useful biomarkers for cancer detection and diagnosis as well as possible therapeutic targets for cancer treatment.

Expression profiling of claudins in the human gastrointestinal tract in health and during inflammatory bowel disease

Background. Claudins, being part of the tight junction protein family, partially determine the integrity and paracellular permeability of the intestinal epithelium. The aim of this study was twofold. First, the authors set out to create an overview of claudin mRNA expression along the proximal-distal axis of the healthy human intestine. Second, the authors aimed to analyze expression levels of claudins in patients with active and inactive inflammatory bowel diseases (IBD) such as Crohn's disease or ulcerative colitis (UC). Methods. mRNA expression levels of claudins were determined in gastrointestinal biopsies from healthy patients as well as patients diagnosed with IBD using SybrGreen real-time PCR. Results. Claudins show distinct expression patterns throughout the gastrointestinal tract. Some claudins show a proximal expression pattern, such as CLDN18 which is solely expressed in the stomach, and CLDN2 and -15 that are predominantly expressed in the proximal parts of the gastrointestinal tract. Other claudins, such as CLDN3, -4, -7 and -8, are predominantly expressed in the distal parts of the gastrointestinal tract or show a ubiquitous expression pattern throughout the entire gastrointestinal tract, which is the case for CLDN12. In addition, we show that changes in claudin expression in IBD are dependent on gastrointestinal location and inflammatory activity. Conclusions. This study provides detailed mRNA expression patterns of various claudins throughout the human gastrointestinal tract. Analysis of expression levels of claudins in patients with CD, active and inactive UC shows that changes in expression are confined to specific intestinal segments and strongly depend on inflammatory activity.

Sequential biphasic changes in claudin1 and claudin4 expression are correlated to colorectal cancer progression and liver metastasis

Terminal progression of colorectal cancer (CRC) culminates in liver metastasis. To identify genes that are involved in the metastatic phenotype, cDNA microarrays were used to analyse mRNA expression profiles of colorectal carcinoma (CC)531 rat colon adenocarcinoma cells for changes related to their homing into the liver. Briefly, CC531 cells were intraportally implanted into the liver of Wag-Rij rats and re-isolated after 3, 6, 9, 14 and 21 days. Compared to control CC531 cells, claudin1 and claudin4 were among the ≥8-fold initially down-regulated genes. The co-culture of tumour cells with isolated rat hepatocytes and Kupffer cells did not induce down-regulation of either claudin1 or 4. When the environment effective on circulating tumour cells was simulated by cell culture conditions favouring their adhesion, only claudin4 showed augmented expression. Knockdown of claudin1 and claudin4 mediated by small interfering RNA caused significantly increased migration and decreased clonogenic growth of tumour cells (P < 0.05), but had no effect on their proliferation. These experimental results were paralleled by increased claudin1 and claudin4 expression in human CRC samples in Union for International Cancer Control (UICC) stages I–III, as evaluated by real-time PCR. Increased claudin4 levels were correlated with significantly reduced overall survival (log-rank test, P= 0.018). Further, significantly (P < 0.05) reduced expression of claudin1 and claudin4 was observed in stage IV and liver metastasis by immunohistochemistry. In conclusion, sequential biphasic changes in claudin1 and claudin4 expression occur during the homing of rat CC531 CRC cells to the liver. This modulation is reflected by significant changes in claudin expression in human primary and metastatic CRC.

Methods to Examine Tight Junction Physiology in Cancer Stem Cells: TEER, Paracellular Permeability, and Dilution Potential Measurements

Our understanding of the essential role played by cancer stem cells or tumor-initiating cells in epithelial cell-derived tumor types is rapidly advancing. Nevertheless, the identification and characterization of these cells pose a considerable challenge. Among changes in the epithelium in oncogenesis are changes in the permeability barrier, a phenotypic trait based on tight junction formation and function. Tight junctions regulate the movement of solutes, ions and water across the paracellular space. On a cellular level, they maintain cell polarity by limiting the lateral diffusion of membrane components. Depending on the type of epithelial tissue, the barrier characteristics with respect to electrical resistance, size and ion charge selectivity vary quite significantly. Thus, elucidating changes in expression of Claudins, an essential component of tight junctions, has become a very active area of investigation in oncogenesis. This chapter provides detailed protocols on how to quantify three aspects of tight junction physiology using in vitro cell culture systems that are particularly applicable to analysis and comparison of cancer stem cells and their normal counterparts.

Tight Junction Expression Reflects Changes in Intestinal Permeability in the Developing Mouse Intestine

Necrotizing Enterocolitis (NEC) is a devastating disease of preterm infants. Immature intestinal barrier function has been implicated in its pathogenesis. Tight junctions (TJ), comprised of transmembrane Claudins which seal the paracellular space, are a vital component of the intestinal barrier and its regulation of intestinal permeability. However, little is known about how TJ development in the immature intestine influences changes in intestinal permeability. We screened mRNA expression of 29 TJ proteins by qRT‐PCR in the ileum of developing mice over the first 8 weeks of life. Selective TJ proteins were further analyzed by immunoblot analysis and localized by immunofluorescence labeling/confocal microscopy (IF). Temporal changes in expression were correlated with FITC‐dextran intestinal permeability assays. Most TJ proteins demonstrated decreased expression over time. However, Claudin‐3 and Claudin‐7 expression peaked at 3 weeks. IF demonstrated appropriate localization of Claudins in TJ after 3 weeks. Over the same period, intestinal permeability decreased. Changes in Claudin expression likely drive changes in intestinal permeability by tightening the paracellular space. Further studies are needed to elucidate what factors influence temporal changes in TJ expression and intestinal permeability and how they may affect predisposition to NEC.

Effect of claudins 6 and 9 on paracellular permeability in MDCK II cells.

The neonatal proximal tubule has a lower permeability to chloride, higher resistance, and higher relative sodium-to-chloride permeability (P(Na)/P(Cl)) than the adult tubule, which may be due to maturational changes in the tight junction. Claudins are tight-junction proteins between epithelial cells that determine paracellular permeability characteristics of epithelia. We have previously described the presence of two claudin isoforms, claudins 6 and 9, in the neonatal proximal tubule and subsequent reduction of these claudins during postnatal maturation. The question is whether changes in claudin expression are related to changes in functional characteristics in the neonatal tubule. We transfected claudins 6 and 9 into Madin-Darby canine kidney II (MDCK II) cells and performed electrophysiological studies to determine the resultant changes in physiological characteristics of the cells. Expression of claudins 6 and 9 resulted in an increased transepithelial resistance, decreased chloride permeability, and decreased P(Na)/P(Cl) and P(HCO3)/P(Cl). These findings constitute the first characterization of the permeability characteristics of claudins 6 and 9 in a cell model and may explain why the neonatal proximal tubule has lower permeability to chloride and higher resistance than the adult proximal tubule.

Decreased expression of claudin-1 is correlated with recurrence status in esophageal squamous cell carcinoma

Claudins are transmembrane proteins and major constitutes of tight junctions, and participate in the paracellular barrior and cellular connecting functions. They have been shown to be differentially regulated in malignant tumors and play a role in carcinogenesis and tumor progression. Recent studies have shown changes in expression of claudins during tumorigenesis. However, a causal relationship between claudin expression and cancer recurrent status has not been established. In this study, we examined 54 esophageal cancer cases to assess immunohistochemical expression patterns of claudin-1. Eleven (20.4%) of 54 cases had negative immunostaining for claudin-1. Decreased expression of claudin-1 was statistically correlated with recurrence status (P = 0.018). The cases with lymphatic vessel permeation showed reduced expression patterns of claudin-1 (P = 0.033). Decreased expression of claudin-1 was also correlated with short diseasefree survival (P = 0.0003) and overall survival (P = 0.0045). The results indicated that claudin-1 expression was correlated with the recurrence status and poor prognosis in esophageal cancer and claudin-1 expression may be a good indicator of recurrence in esophageal cancer.

Chronic alcohol ingestion alters claudin expression in the alveolar epithelium of rats

Previously we determined that chronic alcohol ingestion (6 weeks) in rats increases lung epithelial permeability in vivo ∼5–6-fold and promotes flooding of the alveolar airspaces with proteinaceous fluid in response to stresses such as sepsis. In parallel, alveolar epithelial cells isolated from alcohol-fed rats fail to form tight monolayers in vitro, even when cultured for up to 8 days in the absence of alcohol. However, the molecular mechanisms underlying alcohol-induced permeability are unknown. Claudins are key components of tight junctions that restrict the paracellular movement of water, proteins, and solutes across cellular barriers including the alveolar epithelium. In this study, we examined the expression of multiple members of the claudin protein family in the lungs of alcohol-fed versus control-fed rats (Lieber–DeCarli liquid diet with either 36% of calories as alcohol or an isocaloric substitution with maltin-dextrin for 6 weeks). We determined that chronic alcohol ingestion affected the expression of multiple claudins; most striking were decreases in claudin-1 and claudin-7, and an increase in claudin-5, in the whole lung and in alveolar epithelial monolayers derived from alcohol-fed rats. In parallel, immunocytochemistry of alveolar epithelial monolayers from alcohol-fed rats revealed abnormal intracellular accumulation of claudin-7 protein and relatively decreased localization to cell membranes. Claudin-1 and claudin-7 are relatively specific to alveolar epithelial type I pneumocytes that form the vast majority of the alveolar epithelial barrier in vivo, and increases in claudin-5 have been associated with increased epithelial permeability in other systems. Therefore, these findings suggest that changes in claudin expression in the alveolar epithelium produce a “leakier” phenotype that renders the alcoholic lung susceptible to alveolar flooding during acute inflammatory stresses.

Altered claudin expression is a feature of chronic plaque psoriasis

Epithelial tight junctions play a central role in cell-cell adhesion and are necessary for the selective paracellular movement of ions. Claudins are key components of tight junctions and their expression is altered in gut epithelia in a variety of inflammatory enteropathies, including ulcerative colitis and Crohn's disease. Psoriasis is a chronic inflammatory skin disease affecting approximately 2% of the western population, with significantly increased occurrence in individuals with Crohn's disease. Initial studies investigated the expression of claudins in skin of healthy volunteers and patients with chronic plaque psoriasis. We report here that claudins-1 and -3 are the major protein species present in the epidermis of healthy skin; they are expressed on the surface of epidermal keratinocytes, consistent with their localization to tight junctions. In plaques of psoriasis, claudin-1 was not identifiable in the epidermis, although typical staining patterns were observed in clinically normal, uninvolved skin of patients with psoriasis. Claudin-3 was present in the epidermal granular cell layer in normal skin, but was only identified within the cytosol of epidermal keratinocytes in both involved and uninvolved skin of psoriasis patients. We examined further whether exposure of keratinocytes in vitro to pro-inflammatory cytokines mimicked the observed changes in claudin expression seen in chronic plaque psoriasis; lipopolysaccharide, interferon-gamma and tumour necrosis factor-alpha had no effect on claudin protein expression or distribution. Addition of interleukin-1beta, however, resulted in down-regulation of claudins-1 and -3. Tumour necrosis factor-alpha and interleukin-1beta were further used in an in vivo model of skin inflammation; interleukin-1beta alone modulated claudin protein expression in this system. These data demonstrate that epidermal claudin expression is altered in chronic plaque psoriasis and that expression is in part modulated by interleukin-1beta.

Effects of transdifferentiation and EGF on claudin isoform expression in alveolar epithelial cells.

Rat alveolar epithelial type II cells grown on polycarbonate filters form high-resistance monolayers and concurrently acquire many phenotypic properties of type I cells. Treatment with EGF has previously been shown to increase transepithelial resistance across alveolar epithelial cell (AEC) monolayers. We investigated changes in claudin expression in primary cultured AEC during transdifferentiation to the type I cell-like phenotype (days 0, 1, and 8), and on day 5 in culture +/- EGF (10 ng/ml) from day 0 or day 4. Claudins 4 and 7 were increased, whereas claudins 3 and 5 were decreased, on later compared with earlier days in culture. Exposure to EGF led to increases in claudins 4 and 7 and decreases in claudins 3 and 5. Claudin 1 was only faintly detectable in freshly isolated type II cells and remained unchanged over time in culture and after exposure to EGF. These results suggest that increases in transepithelial resistance accompanying AEC transdifferentiation and/or EGF exposure are mediated, at least in part, by changes in the pattern of expression of specific claudin isoforms.

Epidermal Growth Factor Receptor Activation Differentially Regulates Claudin Expression and Enhances Transepithelial Resistance in Madin-Darby Canine Kidney Cells

Tight junctions (TJs) are the most apical cell-cell junctions, and claudins, the recently identified TJ proteins, are critical for maintaining cell-cell adhesion in epithelial cell sheets. Based on their in vivo distribution and the results of overexpression studies, certain claudins, including claudin-1 and -4, are postulated to increase, whereas other claudins, especially claudin-2, are postulated to decrease the overall transcellular resistance. The overall ratio among claudins expressed in a cell/tissue has been hypothesized to define the complexity of TJs. Disruption of the TJs contributes to various human diseases, and a correlation between reduction of TJ function and tumor dedifferentiation has been postulated. The epidermal growth factor (EGF) receptor (EGFR) is overexpressed in a wide spectrum of epithelial cancers, and its expression correlates with a more metastatic cancer phenotype. However, normal functioning of EGFR is essential for normal epithelial cell proliferation and differentiation. The role of EGFR-dependent signaling in the development and maintenance of epithelial TJ integrity has not been studied in detail. This study demonstrates that, in polarized Madin-Darby canine kidney II cells, EGF-induced EGFR activation significantly inhibited claudin-2 expression while simultaneously inducing cellular redistribution and increased expression of claudin-1, -3, and -4. Accompanying these EGF-induced changes in claudin expression was a 3-fold increase in transepithelial resistance, a functional measure of TJs. In contrast, there were no alterations in protein expression and/or intracellular localization of other TJ-related proteins (ZO-1 and occludin) or adherens junction-associated proteins (E-cadherin and beta-catenin), suggesting that EGF regulates TJ function through selective and differential regulation of claudins.