Heterogeneity in expression and subcellular localization of tight junction proteins, claudin-10 and -15, examined by RT-PCR and immunofluorescence microscopy

Tight junctions regulate paracellular permeability, create the luminal fluid microenvironment of blood vessels and the digestive tract, and also form the protective barrier in the stratified epithelium including the epidermis. Claudins are the integral membrane proteins at tight junctions and form a multigene family composed of at least 24 members, but knowledge of the subcellular localization of each claudin is still fragmentary. We performed RT-PCR for fifteen claudin species to examine the mRNA expression in various mouse tissues, and focused on investigating the subcellular localization of claudin-10 and -15 by immunofluorescence microscopy in various rat tissues. Neither claudin-10 nor -15 was detected in vascular endothelial cells in most tissues, and these claudins were restricted to the vasa recta in the kidney medulla. Both claudins were also detected at apical tight junctions in the epithelium of the jejunum with no intensity gradients along the crypt-to-villus axis. However, both claudins were expressed only in the basal half of the crypt epithelium in the colon, showing obvious gradients along crypt-to-surface axis. Moreover, claudin-10 showed the ectopic subcellular localization where tight junction strands do not exist. Claudin-10 was detected along the entire lateral membranes of acinar cells in addition to the apical tight junctions in exocrine glands, and in the cytoplasm of basal cells in the stratified epithelium including the dorsal skin and cutaneous stomach. These heterogeneous distributions of claudin-10 and -15 in tissues may be related to the differences in paracellular permeability among tissues.     

Intercellular junctions in Ewing sarcoma/primitive neuroectodermal tumor: additional evidence of epithelial differentiation.

Ewing sarcoma/primitive neuroectodermal tumor (ES/PNET) has recently been shown to frequently express cytokeratins, suggesting partial epithelial differentiation. Older ultrastructural studies have documented primitive cell-cell junctions in ES/PNET, reportedly resembling poorly formed desmosomes. Recently, paraffin-reactive antibodies have become available to proteins found in a variety of intercellular junctions indicative of epithelial differentiation, including tight junctions, desmosomes and adherens junctions. We examined intercellular junction protein expression in a large number of genetically confirmed ES/PNET. Formalin-fixed, paraffin-embedded sections from 23 primary and seven recurrent or metastatic cases of genetically confirmed ES/PNET were immunostained for claudin-1 and occludin (tight junction structural proteins), zonula occludens-1 (ZO-1, tight junction linker protein), desmoglein 1/2 (desmosomal adherens protein), desmoplakin (desmosomal structural protein) and E-cadherin (epithelial adherens junction protein), using steam heat-induced epitope retrieval and the Dako Envision system. Cases with >5% positive cells were scored as 'positive'. Normal colonic epithelium and skin served as external positive controls. Claudin-1 was expressed by 19 of 30 specimens (63%), ZO-1 was expressed by 15 of 29 specimens (51%), and occludin was expressed by three of 28 specimens (11%). In 28 specimens all three tight junction markers were evaluable. In all, 15 samples (54%) expressed only one tight junction marker, and 10 samples (36%) expressed two tight junction markers. No case expressed all three tight junction markers. Desmoglein was expressed in one of 30 (3%) samples. Desmoplakin was expressed in two of 28 (7%) samples. E-cadherin was negative in all cases. Our data suggest that many of the previously described cell-cell junctions in ES/PNET are poorly formed tight junctions, given the high frequency of claudin-1 and ZO-1 expression. This may underestimate the true frequency of tight junction protein expression in ES/PNET, as there are at least 20 different claudins and other ZO proteins. These tight junctions are almost certainly abnormal, given the absence of occludin expression in most cases. Desmosomal and adherens junction protein expression was rare to absent. Our findings provide additional evidence that ES/PNET frequently show partial epithelial differentiation.     

Claudins: unlocking the code to tight junction function during embryogenesis and in disease

Gupta IR, Ryan AK. Claudins: unlocking the code to tight junction function during embryogenesis and in disease. Claudins are the structural and molecular building blocks of tight junctions. Individual cells express more than one claudin family member, which suggests that a combinatorial claudin code that imparts flexibility and dynamic regulation of tight junction function could exist. Although we have learned much from manipulating claudin expression and function in cell lines, loss‐of‐function and gain‐of‐function experiments in animal model systems are essential for understanding how claudin‐based boundaries function in the context of a living embryo and/or tissue. These in vivo manipulations have pointed to roles for claudins in maintaining the epithelial integrity of cell layers, establishing micro‐environments and contributing to the overall shape of an embryo or tissue. In addition, loss‐of‐function mutations in combination with the characterization of mutations in human disease have demonstrated the importance of claudins in regulating paracellular transport of solutes and water during normal physiological states. In this review, we will discuss specific examples of in vivo studies that illustrate the function of claudin family members during development and in disease.     

Decreased distribution of lung epithelial junction proteins after intratracheal antigen or lipopolysaccharide challenge: correlation with neutrophil influx and levels of BALF sE-cadherin

Distribution of airway junctional complex proteins after antigen or lipopolysaccharide challenge in sensitized or naive mice, respectively, was investigated. E-cadherin immunoreactivity was detected continuously along neighboring epithelial cell borders and between adjacent alveolar epithelial cells in naive and saline-challenged mice. Occludin and ZO-1 immunoreactivity were observed in the tight junction areas. Both challenges induced changes in epithelial morphology and phenotype, accompanied initially by focal loss of epithelial E-cadherin that increased in size with time and number of allergen challenges. Allergen challenge also led to focal loss of occludin and ZO-1. Western blot analysis revealed increased levels of sE-cadherin in lavage fluid after either challenge, and this increase correlated with lavage neutrophil numbers (P = 0.002). Immunocytochemistry of lavage cells 6 h after either challenge revealed E-cadherin epitopes within cytoplasmic vacuoles of neutrophils, the major cell type. In contrast, peripheral blood neutrophils or tissue neutrophils before epithelial transmigration were negative, suggesting that in airway inflammation, E-cadherin extracellular domain is cleaved by neutrophils during epithelial penetration, instigating the destabilization of adherens and tight junctions. This junctional deterioration could lead to a progressive decrease in epithelial integrity and induce alterations in epithelial morphology, with consequent enhanced paracellular transit of antigens and pathogens.     

Claudins: control of barrier function and regulation in response to oxidant stress

Claudins are a family of nearly two dozen transmembrane proteins that are a key part of the tight junction barrier that regulates solute movement across polarized epithelia. Claudin family members interact with each other, as well as with other transmembrane tight junction proteins (such as occludin) and cytosolic scaffolding proteins (such as zonula occludens-1 (ZO-1)). Although the interplay between all of these different classes of proteins is critical for tight junction formation and function, claudin family proteins are directly responsible for forming the equivalent of paracellular ion selective channels (or pores) with specific permeability and thus are essential for barrier function. In this review, we summarize current progress in identifying structural elements of claudins that regulate their transport, assembly, and function. The effects of oxidant stress on claudins are also examined, with particular emphasis on lung epithelial barrier function and oxidant stress induced by chronic alcohol abuse.     

Apically exposed, tight junction-associated beta1-integrins allow binding and YopE-mediated perturbation of epithelial barriers by wild-type Yersinia bacteria

Using polarized epithelial cells, primarily MDCK-1, we assessed the mode of binding and effects on epithelial cell structure and permeability of Yersinia pseudotuberculosis yadA-deficient mutants. Initially, all bacteria except the invasin-deficient (inv) mutant adhered apically to the tight junction areas. These contact points of adjacent cells displayed beta1-integrins together with tight junction-associated ZO-1 and occludin proteins. Indeed, beta1-integrin expression was maximal in the tight junction area and then gradually decreased along the basolateral membranes. Wild-type bacteria also opened gradually the tight junction to paracellular permeation of different-sized markers, viz., 20-, 40-, and 70-kDa dextrans and 45-kDa ovalbumin, as well as to their own translocation between adjacent cells in intimate contact with beta1-integrins. The effects on the epithelial cells and their barrier properties could primarily be attributed to expression of the Yersinia outer membrane protein YopE, as the yopE mutant bound but caused no cytotoxicity. Moreover, the apical structure of filamentous actin (F-actin) was disturbed and tight junction-associated proteins (ZO-1 and occludin) were dispersed along the basolateral membranes. It is concluded that the Yersinia bacteria attach to beta1-integrins at tight junctions. Via this localized injection of YopE, they perturb the F-actin structure and distribution of proteins forming and regulating tight junctions. Thereby they promote paracellular translocation of bacteria and soluble compounds.     

Tight junctions and human diseases

Tight junctions are intercellular junctions adjacent to the apical end of the lateral membrane surface. They have two functions, the barrier (or gate) function and the fence function. The barrier function of tight junctions regulates the passage of ions, water, and various macromolecules, even of cancer cells, through paracellular spaces. The barrier function is thus relevant to edema, jaundice, diarrhea, and blood-borne metastasis. On the other hand, the fence function maintains cell polarity. In other words, tight junctions work as a fence to prevent intermixing of molecules in the apical membrane with those in the lateral membrane. This function is deeply involved in cancer cell biology, in terms of loss of cell polarity. Of the proteins comprising tight junctions, integral membrane proteins occludin, claudins, and JAMs have been recently discovered. Of these molecules, claudins are exclusively responsible for the formation of tight-junction strands and are connected with the actin cytoskeleton mediated by ZO-1. Thus, both functions of tight junctions are dependent on the integrity of the actin cytoskeleton as well as ATP. Mutations in the claudin14 and the claudin16 genes result in hereditary deafness and hereditary hypomagnesemia, respectively. Some pathogenic bacteria and viruses target and affect the tight-junction function, leading to diseases. In this review, the relationship between tight junctions and human diseases is summarized.     

Redistribution of Tight Junction Proteins During EPEC Infection In Vivo

Enteropathogenic Escherichia coli (EPEC) is a leading cause of diarrhea among infants. Tight junction plays a vital role in intestinal paracellular permeability by forming physical intercellular barriers in epithelial cells. However, the impact of this enteric pathogen on tight junctions in vivo has not been fully investigated. In the present study, the alterations in tight junctions following EPEC infection in vivo were investigated. Western blot analysis revealed that the tight junction proteins, occludin and claudin-1, were displaced from tight junction membrane microdomains to Triton X-100 soluble fractions after EPEC infection. Changes in intestinal paracellular permeability were determined using the molecular tracer biotin, which was observed to penetrate the epithelia and extended into the lamina propria, indicating disruption in tight junction barrier function. Our results suggested that redistribution of tight junction proteins plays an important role in the disruption of epithelial barrier function induced by EPEC infection, which may provide new insight into the pathogenesis of diarrhea caused by EPEC.     

Neutrophil Transmigration in Inflammatory Bowel Disease Is Associated with Differential Expression of Epithelial Intercellular Junction Proteins

Inflammatory bowel disease (IBD) consisting of ulcerative colitis (UC) and Crohn's (CD) typically displays a waxing and waning course punctuated by disease flares that are characterized by transepithelial migration of neutrophils (PMN) and altered barrier function. Since epithelial barrier function is primarily regulated by the apical most intercellular junction referred to as the tight junction (TJ), our aim was to examine expression of TJ and adherens junction (AJ) proteins in relation to PMN infiltration in mucosal tissue samples from patients with active IBD. Expression of epithelial intercellular TJ proteins (occludin, ZO-1, claudin-1, and JAM) and subjacent AJ (beta-catenin and E-cadherin) proteins were examined by immunoflourescence/confocal microscopy, immunohistochemistry, and Western blotting. Colonic mucosa from patients with UC revealed dramatic, global down-regulation of the key TJ transmembrane protein occludin in regions of actively transmigrating PMN and in quiescent areas in the biopsy samples. Significant decreases in occludin expression were observed at the protein and mRNA levels by Western and Northern blotting. In contrast, expression of other TJ and AJ proteins such as ZO-1, claudin-1, JAM, beta-catenin, and E-cadherin were down-regulated only in epithelial cells immediately adjacent to transmigrating PMN. Analysis of inflamed mucosa from Crohn's disease patients mirrored the results obtained with UC patients. No change in TJ and AJ protein expression was observed in colonic epithelium from patients with collagenous colitis or lymphocytic colitis that are respectively characterized by a thickened subepithelial collagen plate and increased intraepithelial lymphocytes. These results suggest that occludin expression is diminished in IBD by mechanisms distinct from those regulating expression of other intercellular junction proteins. We speculate that down-regulation of epithelial occludin may play a role in enhanced paracellular permeability and PMN transmigration that is observed in active inflammatory bowel disease.     

The association of gap junctions with large particles in the crypt epithelium of the rat small intestine

Intercellular junctions in the epithelium of the rat small intestine were studied by the freeze-fracture method. Gap junctions were found between columnar cells of the crypt far more frequently than between absorptive cells of the villus. In addition, in the crypt, large particles 11 to 13 nm in diameter were often associated with the gap junction which primarily consisted of usual gap junctional particles 8 to 9 nm in diameter. Both the usual and large particles possessed a central pit. The intestinal crypt is the site of epithelial cell proliferation and differentiation in the process of physiological cell renewal. The predominant presence of gap junctions in this region suggests that they play a certain role in the proliferation and differentiation of crypt columnar cells. Further studies are required to elucidate the nature of the large particles, a precursor of typical connexons and/or a functionally different gap junction.     

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.     

紧密连接蛋白claudins与肿瘤的研究进展

Claudins是新近发现的构成细胞紧密连接的一类完整的跨膜蛋白,维持紧密连接特有的栅栏功能和屏障功能。Claudins表达异常与肿瘤的发生发展关系密切。多种途径可能参与了claudins表达的调节。Claudins为肿瘤的诊断和治疗提供了一种新思路。     

Claudins 1, 2, 3, 4, 5 and 7 in solar keratosis and squamocellular carcinoma of the skin

Claudins are tight junction proteins regulating the paracellular permeability of cell layers. We investigated the expression of claudins 1, 2, 3, 4, 5 and 7 in a sample set consisting of a total of 93 cases representing normal skin, actinic keratoses and squamous cell carcinomas of the skin. There were several changes found in claudin expression. Claudin 1 appeared to be progressively decreased in solar keratosis and skin squamous cell carcinomas compared to normal skin while expression of claudin 2 was increased. With claudins 3 and 5 occasional immunoreactivity was found in squamous cell carcinomas. Claudins 4 and 7 were variably expressed in skin neoplasia compared to normal skin. According to the results expression of claudins 1 and 2 change in parallel with the severity of the epidermal preneoplastic and neoplastic lesions thus probably influencing the disturbed epithelial polarity characteristic of these lesions. Claudin 1 under- and claudin 2 overexpression also lead to a leakier epithelial barrier function of the skin with a resulting damage to skin epithelial resistance. Other claudins investigated in this study did not show progressive changes even though occasional overexpression of them was found in skin squamous cell carcinoma.     

Tight junctions in lung cancer and lung metastasis: a review

Tight junctions are structures located in the apicobasal region of the cell membranes. They regulate paracellular solute and electrical permeability of cell layers. Additionally, they influence cellular polarity, form a paracellular fence to molecules and pathogens and divide the cell membranes to apical and lateral compartments. Tight junctions adhere to the corresponding ones of neighbouring cells and by this way also mediate attachment of the cells to one other. Molecules forming the membranous part of tight junctions include occludin, claudins, tricellulin and junctional adhesion molecules. These molecules are attached to scaffolding proteins such as ZO-1, ZO-2 and ZO-3 through which signals are mediated to the cell interior. Expression of tight junction proteins, such as claudins, may be up- or downregulated in cancer and they are involved in EMT thus influencing tumor spread. Like in tumors of other sites, lung tumors show changes in the expression in tight junction proteins. In this review the significance of tight junctions and its proteins in lung cancer is discussed with a focus on the proteins forming the membranous part of these structures.     

Probiotic bacteria induce maturation of intestinal claudin 3 expression and barrier function

An immature intestinal epithelial barrier may predispose infants and children to many intestinal inflammatory diseases, such as infectious enteritis, inflammatory bowel disease, and necrotizing enterocolitis. Understanding the factors that regulate gut barrier maturation may yield insight into strategies to prevent these intestinal diseases. The claudin family of tight junction proteins plays an important role in regulating epithelial paracellular permeability. Previous reports demonstrate that rodent intestinal barrier function matures during the first 3 weeks of life. We show that murine paracellular permeability markedly decreases during postnatal maturation, with the most significant change occurring between 2 and 3 weeks. Here we report for the first time that commensal bacterial colonization induces intestinal barrier function maturation by promoting claudin 3 expression. Neonatal mice raised on antibiotics or lacking the toll-like receptor adaptor protein MyD88 exhibit impaired barrier function and decreased claudin 3 expression. Furthermore, enteral administration of either live or heat-killed preparations of the probiotic Lactobacillus rhamnosus GG accelerates intestinal barrier maturation and induces claudin 3 expression. However, live Lactobacillus rhamnosus GG increases mortality. Taken together, these results support a vital role for intestinal flora in the maturation of intestinal barrier function. Probiotics may prevent intestinal inflammatory diseases by regulating intestinal tight junction protein expression and barrier function. The use of heat-killed probiotics may provide therapeutic benefit while minimizing adverse effects.     

Clostridium difficile toxins disrupt epithelial barrier function by altering membrane microdomain localization of tight junction proteins

The anaerobic bacterium Clostridium difficile is the etiologic agent of pseudomembranous colitis. C. difficile toxins TcdA and TcdB are UDP-glucosyltransferases that monoglucosylate and thereby inactivate the Rho family of GTPases (W. P. Ciesla, Jr., and D. A. Bobak, J. Biol. Chem. 273:16021-16026, 1998). We utilized purified reference toxins of C. difficile, TcdA-10463 (TcdA) and TcdB-10463 (TcdB), and a model intestinal epithelial cell line to characterize their influence on tight-junction (TJ) organization and hence to analyze the mechanisms by which they contribute to the enhanced paracellular permeability and disease pathophysiology of pseudomembranous colitis. The increase in paracellular permeability induced by TcdA and TcdB was associated with disorganization of apical and basal F-actin. F-actin restructuring was paralleled by dissociation of occludin, ZO-1, and ZO-2 from the lateral TJ membrane without influencing the subjacent adherens junction protein, E-cadherin. In addition, we observed decreased association of actin with the TJ cytoplasmic plaque protein ZO-1. Differential detergent extraction and fractionation in sucrose density gradients revealed TcdB-induced redistribution of occludin and ZO-1 from detergent-insoluble fractions constituting "raft-like" membrane microdomains, suggesting an important role of Rho proteins in maintaining the association of TJ proteins with such microdomains. These toxin-mediated effects on actin and TJ structure provide a mechanism for early events in the pathophysiology of pseudomembranous colitis.     

紧密连接蛋白claudins及其在肿瘤发病中的作用

紧密连接分子由occludin，claudins和连接黏附分子(JAMs)3种完整的膜蛋白和闭合小环蛋白(ZO-1，ZO-2和ZO-3)等外周胞浆蛋白组成。Claudin蛋白是构成紧密连接的最主要的功能分子，可维持紧密连接特有的栅栏功能和屏障功能。多种信号分子参与了claudins功能的调节。 Claudins分布于皮肤、脑、神经系统和内脏组织中，其表达有组织特异性，但大多数组织可表达多种claudin蛋白。 Claudin及其结合的蛋白结构改变可导致肿瘤等多种疾病的发生。不同claudins家族成员在不同肿瘤中表达各异,但在恶性肿瘤组织中的表达无论高或低，最终总会引起紧密连接分子的正常结构破坏、生理功能障碍。 claudin蛋白可作?煌琢龅恼锒霞爸瘟频姆肿颖曛尽Ｋ孀欧肿由镅Ъ际醯姆伤俜⒄购投詂laudin更全面的认识，claudin在疾病的检测、诊断、治疗及预后判断方面中会有一个新的应用前景。     

Enteropathogenic Escherichia coli decreases the transepithelial electrical resistance of polarized epithelial monolayers.

The mechanisms whereby enteropathogenic Escherichia coli (EPEC) causes diarrhea remain undefined. We found that EPEC caused a decrease in transepithelial electrical resistance across polarized monolayers of Caco-2 and MDCK epithelial cells. This occurred approximately 6 to 10 h after bacterial addition and was reversible if the monolayers were treated with tetracycline or gentamicin. Although significant alterations in host actin occurred beneath adherent EPEC, actin filaments supporting tight junctions were not noticeably affected in the epithelial cells, nor was the distribution of ZO-1, a tight junction protein. Despite the decrease in transepithelial electrical resistance, EPEC did not cause an increase in [3H]inulin penetration across MDCK monolayers. Unlike in the parental strain, mutations in any loci involved in adherence or formation of attaching and effacing lesions were unable to cause a decrease in transepithelial resistance. These data indicate that EPEC causes a decrease in transepithelial electrical resistance by disrupting a transcellular (intracellular) pathway rather than by disrupting intercellular tight junctions (paracellular) and that these disruptions occur only when attaching and effacing lesions are formed.