Claudin Subtypes Research Articles

Reconstitution of functional tight junctions with individual claudin subtypes in epithelial cells.

The claudin family of membrane proteins is responsible for the backbone structure and function of tight junctions (TJs), which regulate the paracellular permeability of epithelia. It is thought that each claudin subtype has its own unique function and the combination of expressed subtypes determines the permeability property of each epithelium. However, many issues remain unsolved in regard to claudin functions, including the detailed functional differences between claudin subtypes and the effect of the combinations of specific claudin subtypes on the structure and function of TJs. To address these issues, it would be useful to have a way of reconstituting TJs containing only the claudin subtype(s) of interest in epithelial cells. In this study, we attempted to reconstitute TJs of individual claudin subtypes in TJ-deficient MDCK cells, designated as claudin quinKO cells, which were previously established from MDCK II cells by deleting the genes of claudin-1, -2, -3, -4, and -7. Exogenous expression of each of claudin-1, -2, -3, -4, and -7 in claudin quinKO cells resulted in the reconstitution of functional TJs. These TJs did not contain claudin-12 and -16, which are endogenously expressed in claudin quinKO cells. Furthermore, overexpression of neither claudin-12 nor claudin-16 resulted in the reconstitution of TJs, demonstrating the existence of claudin subtypes lacking TJ-forming activity in epithelial cells. Exogenous expression of the channel-forming claudin-2, -10a, -10b, and -15 reconstituted TJs with reported paracellular channel properties, demonstrating that these claudin subtypes form paracellular channels by themselves without interaction with other subtypes. Thus, the reconstitution of TJs in claudin quinKO cells is advantageous for further investigation of claudin functions.Key words: tight junction, claudin, paracellular permeability, epithelial barrier.

Strengthening of the barrier function in human telomerase reverse transcription (hTERT) immortalized corneal and conjunctival epithelium by double-stranded RNA

To investigate the response to polyinosinic:polycytidylic acid [poly(I:C)], a double-stranded RNA Toll-like receptor 3 agonist that mimics viral infection, in the barrier function of two established human telomerase reverse transcriptase-immortalized cell lines, termed HCLE for the human corneal-limbal epithelial line and HCjE for the human conjunctival-epithelial line. In this study, HCLE and HCjE cells were used to evaluate the underlying mechanism of epithelial-cell barrier function regulation. Briefly, HCLE and HCjE cells were first cultured on 12-well Transwell® (Corning®) filter-plates, and reverse transcription-polymerase chain reaction, western blotting, and immunohistochemical examinations were then performed to assess tight junction (TJ)-related protein expression and cellular distribution. Next, the barrier function of the cells was measured via transepithelial electrical resistance (TEER) and paracellular molecular flux. The cells were then stimulated with poly(I:C) and the TEER and TJ-related protein expressions were analyzed. Similar to that in in vivo epithelium, the expression of claudin (CLDN) subtypes CLDN-1, -4, and -7 was observed in the HCLE and HCjE cells, and the barrier function in the HCLE cells was tighter than that in the HCjE cells. Post stimulation with poly(I:C), TEER of the HCLE and HCjE cells increased in a dose- and time-dependent manner, the production of TJ-related protein mRNA and CLDN-4 protein were elevated, and the barrier function of the HCLE and HCjE cells increased, thus possibly indicating that the increased barrier function is a defense mechanism against viral infection.

Role of TM3 in claudin-15 strand flexibility: A molecular dynamics study.

Claudins are cell-cell adhesion proteins within tight junctions that connect epithelial cells together. Claudins polymerize into a network of strand-like structures within the membrane of adjoining cells and create ion channels that control paracellular permeability to water and small molecules. Tight junction morphology and barrier function is tissue specific and regulated by claudin subtypes. Here, we present a molecular dynamics study of claudin-15 strands within lipid membranes and the role of a single-point mutation (A134P) on the third transmembrane helix (TM3) of claudin-15 in determining the morphology of the strand. Our results indicate that the A134P mutation significantly affects the lateral flexibility of the strands, increasing the persistence length of claudin-15 strands by a factor of three. Analyses of claudin-claudin contact in our μsecond-long trajectories show that the mutation does not alter the intermolecular contacts (interfaces) between claudins. However, the dynamics and frequency of interfacial contacts are significantly affected. The A134P mutation introduces a kink in TM3 of claudin-15 similar to the one observed in claudin-3 crystal structure. The kink on TM3 skews the rotational flexibility of the claudins in the strands and limits their fluctuation in one direction. This asymmetric movement in the context of the double rows reduces the lateral flexibility of the strand and leads to higher persistence lengths of the mutant.

Assembly of Tight Junction Strands: Claudin-10b and Claudin-3 Form Homo-Tetrameric Building Blocks that Polymerise in a Channel-Independent Manner

Tight junctions regulate paracellular permeability size and charge selectively. Models have been proposed for the molecular architecture of tight junction strands and paracellular channels. However, they are not fully consistent with experimental and structural data. Here, we analysed the architecture of claudin-based tight junction strands and channels by cellular reconstitution of strands, structure-guided mutagenesis, in silico protein docking and oligomer modelling. Prototypic channel- (Cldn10b) and barrier-forming (Cldn3) claudins were analysed. Förster resonance energy transfer (FRET) assays indicated multistep claudin polymerisation, starting with cis-oligomerization specific to the claudin subtype, followed by trans-interaction-triggered cis-polymerisation. Alternative protomer interfaces were modelled in silico and tested by cysteine-mediated crosslinking, confocal- and freeze fracture EM-based analysis of strand formation. The analysed claudin mutants included also mutations causing the HELIX syndrome. The results indicated that protomers in Cldn10b and Cldn3 strands form similar antiparallel double rows, as has been suggested for Cldn15. Mutually stabilising ‐hydrophilic and hydrophobic ‐ cis- and trans-interfaces were identified that contained novel key residues of extracellular segments ECS1 and ECS2.Hydrophobic clustering of the flexible ECS1 β1β2 loops together with ECS2–ECS2 trans-interaction is suggested to be the driving force for conjunction of tetrameric building blocks into claudin polymers. Cldn10b and Cldn3 are indicated to share this polymerisation mechanism. However, in the paracellular centre of tetramers, electrostatic repulsion may lead to formation of pores (Cldn10b) and electrostatic attraction to barriers (Cldn3). Combining in vitro data and in silico modelling, this study improves mechanistic understanding of paracellular permeability regulation by elucidating claudin assembly and its pathologic alteration as in HELIX syndrome.

Enhancement of the thermostability of mouse claudin-3 on complex formation with the carboxyl-terminal region of Clostridium perfringens enterotoxin improves crystal quality.

Tight junctions regulate substance permeation through intercellular spaces as a physical barrier or a paracellular pathway, and play an important role in maintaining the internal environment. Claudins, which are tetraspan-transmembrane proteins, are pivotal components of tight junctions. In mammals 27 claudin subtypes have been identified, each of which interacts with specific subtypes. Although the crystal structures of several subtypes have been determined, the molecular mechanisms underlying subtype specificity remain unclear. Here, mouse claudin-3 (mCldn3) was crystallized in complex with the C-terminal region of Clostridium perfringens enterotoxin (C-CPE) for the structural analysis of an additional claudin subtype. mCldn3 alone was difficult to crystallize, but complex formation with C-CPE enhanced the thermostability of mCldn3 and facilitated its crystallization. The introduction of an S313A mutation into C-CPE further improved its thermostability, and the resolution limits of the diffraction data sets improved from 8 Å for the wild-type complex to 4.7 Å for the S313A mutant complex.

Crystal structures of claudins: insights into their intermolecular interactions.

Claudins are four-transmembrane proteins that constitute the backbone of tight junction strands via self-polymerization in the apicolateral membranes of epithelial cells. Together with their cell-cell adhesion function, claudin proteins form the paracellular barrier and/or channels through epithelial cell sheets whose permeability is primarily dependent on the claudin subtype. Recently determined crystal structures of several claudins revealed the unique claudin fold of four transmembrane helices in a left-handed helical bundle with an extracellular β-sheet domain. Here, we focus on the structural basis of the intermolecular interactions between claudin molecules and between the Clostridium perfringens enterotoxin and its receptor claudins.

Establishment of a Human Blood-Brain Barrier Co-culture Model Mimicking the Neurovascular Unit Using Induced Pluri- and Multipotent Stem Cells.

SummaryIn vitro models of the human blood-brain barrier (BBB) are highly desirable for drug development. This study aims to analyze a set of ten different BBB culture models based on primary cells, human induced pluripotent stem cells (hiPSCs), and multipotent fetal neural stem cells (fNSCs). We systematically investigated the impact of astrocytes, pericytes, and NSCs on hiPSC-derived BBB endothelial cell function and gene expression. The quadruple culture models, based on these four cell types, achieved BBB characteristics including transendothelial electrical resistance (TEER) up to 2,500 Ω cm2 and distinct upregulation of typical BBB genes. A complex in vivo-like tight junction (TJ) network was detected by freeze-fracture and transmission electron microscopy. Treatment with claudin-specific TJ modulators caused TEER decrease, confirming the relevant role of claudin subtypes for paracellular tightness. Drug permeability tests with reference substances were performed and confirmed the suitability of the models for drug transport studies.

Claudin-5, -7, and -18 suppress proliferation mediated by inhibition of phosphorylation of Akt in human lung squamous cell carcinoma

Abnormal expression of claudin (CLDN) subtypes has been reported in various solid cancers. However, it is unknown which subtype plays a key role in the regulation of proliferation in cancer cells. The expression of CLDN3-5, 7, and 18 in human lung squamous carcinoma tissues was lower than that in normal tissue. Here, we examined which combination of exogenous CLDNs expression inhibits proliferation and the molecular mechanism using human lung squamous RERF-LC-AI cells. Real-time polymerase chain reaction and western blotting showed that CLDN3-5, 7, and 18 are little expressed in RERF-LC-AI cells. In the exogenously transfected cells, CLDN5, 7, and 18 were distributed in the cell–cell contact areas concomitant with ZO-1, a tight junctional scaffolding protein, whereas CLDN3 and 4 were not. Cell proliferation was individually and additively suppressed by CLDN5, 7, and 18. The expression of these CLDNs showed no cytotoxicity compared with mock cells. CLDN5, 7, and 18 increased p21 and decreased cyclin D1, resulting in the suppression of cell cycle G1-S transition. The expression of these CLDNs inhibited phosphorylation of Akt without affecting phosphorylated ERK1/2. Furthermore, these CLDNs inhibited the nuclear localization of Akt and its association with 3-phosphoinositide-dependent protein kinase-1 (PDK1). The suppression of G1-S transition caused by CLDN5, 7, and 18 was rescued by the expression of constitutively active-Akt. We suggest that the reduction of CLDN5, 7, and 18 expression loses the suppressive ability of interaction between PDK1 and Akt and causes sustained phosphorylation of Akt, resulting in the disordered proliferation in lung squamous carcinoma cells.

Claudin-18 inhibits cell proliferation and motility mediated by inhibition of phosphorylation of PDK1 and Akt in human lung adenocarcinoma A549 cells

Abnormal expression of claudin subtypes has been reported in various cancers. However, the pathological role of each claudin has not been clarified in detail. Claudin-18 was absent in human non-small cell and small cell lung cancers, although it is expressed in normal lung tissues. Here, we examined the effect of claudin-18 expression on the expression of junctional proteins, cell proliferation, and cell motility using human lung adenocarcinoma A549 cells. Real-time PCR and western blotting showed that exogenous expression of claudin-18 had no effect on the expression of junctional proteins including claudin-1, zonula occludens-1 (ZO-1), occludin, and E-cadherin. Claudin-18 was mainly distributed in cell–cell contact areas concomitant with ZO-1. Cell proliferation was significantly decreased at 48 and 72h after seeding of claudin 18-expressing cells. Claudin-18 suppressed cell motility, whereas it increased cell death in anoikis. Claudin-18 decreased phosphorylated (p)-3-phosphoinositide-dependent protein kinase-1 (PDK1) and p-Akt levels without affecting p-epidermal growth factor receptor and p-phosphatidylinositol-3 kinase (PI3K) levels. Furthermore, claudin-18 was bound with PDK1 and suppressed the nuclear localization of PDK1. We suggest that claudin-18 suppresses the abnormal proliferation and motility of lung epithelial cells mediated by inhibition of the PI3K/PDK1/Akt signaling pathway.

Evidence for a role of claudin 2 as a proximal tubular stress responsive paracellular water channel

Claudins are the major proteins of the tight junctions and the composition of claudin subtypes is decisive for the selective permeability of the paracellular route and thus tissue specific function. Their regulation is complex and subject to interference by several factors, including oxidative stress. Here we show that exposure of cultured human proximal tubule cells (RPTEC/TERT1) to the immunosuppressive drug cyclosporine A (CsA) induces an increase in transepithelial electrical resistance (TEER), a decrease in dome formation (on solid growth supports) and a decrease in water transport (on microporous growth supports). In addition, CsA induced a dramatic decrease in the mRNA for the pore forming claudins -2 and -10, and the main subunits of the Na+/K+ ATPase. Knock down of claudin 2 by shRNA had no discernable effect on TEER or dome formation but severely attenuated apical to basolateral water reabsorption when cultured on microporous filters. Generation of an osmotic gradient in the basolateral compartment rescued water transport in claudin 2 knock down cells. Inhibition of Na+/K+ ATPase with ouabain prevented dome formation in both cell types. Taken together these results provide strong evidence that dome formation is primarily due to transcellular water transport following a solute osmotic gradient. However, in RPTEC/TERT1 cells cultured on filters under iso-osmotic conditions, water transport is primarily paracellular, most likely due to local increases in osmolarity in the intercellular space. In conclusion, this study provides strong evidence that claudin 2 is involved in paracellular water transport and that claudin 2 expression is sensitive to compound induced cellular stress.

Expression and Distribution of Claudin Subtypes in Human Corneal Endothelium

To investigate the expression pattern of claudins in human corneal endothelium, and to evaluate the functional role of the claudin-10b subtype. Corneal endothelium with Descemet's membrane and the corneal epithelium were stripped from donor human corneal stroma. Reverse transcription-polymerase chain reaction (RT-PCR) was performed to evaluate the claudin subtypes expressed in corneal endothelium, stroma, and epithelium. Immunohistochemistry was performed to confirm the expression of claudin subtypes in corneal endothelium, and the expression pattern was compared to that of corneal epithelium. Finally, transendothelial resistance (TER), short-circuit current (SCC), and potential difference (PD) were measured in human corneal endothelial cell line B4G12 cells with or without claudin-10 small interfering RNA (siRNA) transfection by Ussing chamber system. Transcripts for claudin-1, -2, -3, -4, -7, -10b, -11, -15, -22, -23, and -24 were identified in corneal endothelium sample by RT-PCR. Immunohistochemistry confirmed the expression of claudin-1, -2, -4, -7, -10, -11 -15, -22, and -23 in corneal endothelium. In corneal stroma, claudin-1, -2, -3, -4, -5, -6, -7, -8, -10b, -11, -12, -14, -15, -22, -23, and -24 were identified by RT-PCR. In corneal epithelium, claudin-1, -3, -4, -7, -11, -14, and -23 were identified by immunohistochemistry and RT-PCR. Downregulation of claudin-10b by siRNA resulted in the decrease of SCC and PD, but not TER, in B4G12 cells. The expression pattern of claudin-10b(+)/claudin-14(-) was specific in corneal endothelium among the three corneal layers. Claudin-10b may play an important role in the tight junction of corneal endothelium.

Differential expression of claudin-23 in retina of oxygen-induced retinopathy mice

Background Researches showed that some claudin subtypes are closely related to the integrity of blood retina barrier and participate in pathological angiogenesis.Ciaudin-23 is a novel discovered claudin subtype.Understanding its expression in the retina and its relationship with retinal vascular lesion is very important for the targeting treatment of retinal vascular diseases.Objective The aim of this study was to investigate the differential expression of claudin-23 in the retina of mice with oxygen-induced retinopathy(OIR).Methods OIR mice models were established by putting postnatal day 7 (P7)SPF C57BL/6J mice in the enclosed container with oxygen concentration of 75％ ±3％ together with mother mice for 5 days.The age-matched normal mice were used as controls.Fluorescein isothiocyanate dextran (FITC-dextran) was retro-orbitally injected to mice to confirm the models by whole-mount retina.Expressions of claudin-23 mRNA and protein in P12,P17 and P25 mice retinas were examined using real-time PCR and Western blot.lmmunofluorescent staining of frozen section was used to identify the expression and location of claudin-23 in the normal and OIR mice.Results Tortuous and engorged retinal vessels were found in P17 mice retina of the OIR group.Nonperfusion zones,abnormal capillary network and lots of pathological neovascular tufts were observed on the whole-mount retinas.The level of claudin-23 mRNA was slowly increased with the aging,reaching 2.3 fold in P25 normal mice more than P7 mice.However,that in OIR mice demonstrated 12.5-fold elevation from P7 to P25.There were no significant change in the mRNA and protein levels of claudin-23 in P7 mice retina between the normal group and OIR group(P＞0.05).But the mRNA level of claudin-23 in P12,P17 and P25 mice retinas was significantly higher in the OIR group than that in normal group(P＜0.05).The protein level of claudin-23 in P25 mice retina was significantly higher in the OIR group than that in normal group(P＜0.05).Claudin23 expressed mainly in inner nuclear cell layer and retinal ganglion cell layer of the normal mice,but claudin-23 expression was obviously increased in OIR mice.No colocalization of claudin-23 and Isolectin B4 was found in the retina in both normal and OIR mice by double immunofluorescent staining.Conclusions Claudin-23 expression upregulates in the retina of OIR mice.Claudin-23 locates in retinal neurons but not vascular endothelial cells. Key words: Claudin-23; Retina; Oxygen-induced retinopathy

Lipopolysaccharide Disrupts the Milk-Blood Barrier by Modulating Claudins in Mammary Alveolar Tight Junctions

Mastitis, inflammation of the mammary gland, is the most costly common disease in the dairy industry, and is caused by mammary pathogenic bacteria, including Escherichia coli. The bacteria invade the mammary alveolar lumen and disrupt the blood-milk barrier. In normal mammary gland, alveolar epithelial tight junctions (TJs) contribute the blood-milk barrier of alveolar epithelium by blocking the leakage of milk components from the luminal side into the blood serum. In this study, we focused on claudin subtypes that participate in the alveolar epithelial TJs, because the composition of claudins is an important factor that affects TJ permeability. In normal mouse lactating mammary glands, alveolar TJs consist of claudin-3 without claudin-1, -4, and -7. In lipopolysaccharide (LPS)-induced mastitis, alveolar TJs showed 2-staged compositional changes in claudins. First, a qualitative change in claudin-3, presumably caused by phosphorylation and participation of claudin-7 in alveolar TJs, was recognized in parallel with the leakage of fluorescein isothiocyanate-conjugated albumin (FITC-albumin) via the alveolar epithelium. Second, claudin-4 participated in alveolar TJs with claudin-3 and claudin-7 12 h after LPS injection. The partial localization of claudin-1 was also observed by immunostaining. Coinciding with the second change of alveolar TJs, the severe disruption of the blood-milk barrier was recognized by ectopic localization of β-casein and much leakage of FITC-albumin. Furthermore, the localization of toll-like receptor 4 (TLR4) on the luminal side and NFκB activation by LPS was observed in the alveolar epithelial cells. We suggest that the weakening and disruption of the blood-milk barrier are caused by compositional changes of claudins in alveolar epithelial TJs through LPS/TLR4 signaling.

Claudins play a role in normal and tumor cell motility

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

Mechanism of Clostridium perfringens Enterotoxin Interaction with Claudin-3/-4 Protein Suggests Structural Modifications of the Toxin to Target Specific Claudins

Claudins (Cld) are essential constituents of tight junctions. Domain I of Clostridium perfringens enterotoxin (cCPE) binds to the second extracellular loop (ECL2) of a subset of claudins, e.g. Cld3/4 and influences tight junction formation. We aimed to identify interacting interfaces and to alter claudin specificity of cCPE. Mutagenesis, binding assays, and molecular modeling were performed. Mutation-guided ECL2 docking of Cld3/4 onto the crystal structure of cCPE revealed a common orientation of the proposed ECL2 helix-turn-helix motif in the binding cavity of cCPE: residues Leu(150)/Leu(151) of Cld3/4 bind similarly to a hydrophobic pit formed by Tyr(306), Tyr(310), and Tyr(312) of cCPE, and Pro(152)/Ala(153) of Cld3/4 is proposed to bind to a second pit close to Leu(223), Leu(254), and Leu(315). However, sequence variation in ECL2 of these claudins is likely responsible for slightly different conformation in the turn region, which is in line with different cCPE interaction modes of Cld3 and Cld4. Substitutions of other so far not characterized cCPE residues lining the pocket revealed two spatially separated groups of residues (Leu(223), Asp(225), and Arg(227) and Leu(254), lle(258), and Asp(284)), which are involved in binding to Cld3 and Cld4, albeit differently. Involvement of Asn(148) of Cld3 in cCPE binding was confirmed, whereas no evidence for involvement of Lys(156) or Arg(157) was found. We show structure-based alteration of cCPE generating claudin binders, which interact subtype-specific preferentially either with Cld3 or with Cld4. The obtained mutants and mechanistic insights will advance the design of cCPE-based modulators to target specific claudin subtypes related either to paracellular barriers that impede drug delivery or to tumors.

Expression of Claudins and Their Prognostic Significance in Noninvasive Urothelial Neoplasms of the Human Urinary Bladder

The members of the claudin family are major integral transmembrane protein constituents of tight junctions. Normal and neoplastic tissues can be characterized by unique qualitative and quantitative distribution of claudin subtypes, which may be related to clinicopathological features. Differential diagnosis and prognosis of nonmuscle invasive tumor entities of urinary bladder epithelium are often challenging. The aim was to investigate the expression profile of claudins in inverted urothelial papillomas (IUPs), urothelial papillomas (UPs), papillary urothelial neoplasms of low malignant potential (PUNLMPs), and intraepithelial (Ta), low-grade urothelial cell carcinomas (LG-UCCs) in order to reveal potential prognostic and differential diagnostic values of certain claudins. Claudin-1, -2, -4, and -7 protein expressions detected by immunohistochemistry and clinical data were analyzed in 15 IUPs, 20 UPs, 20 PUNLMPs, and 20 LG-UCCs. UPs, PUNLMPs, and LG-UCCs showed significantly decreased claudin-1 expression in comparison to IUPs. LG-UCCs expressing claudin-4 over the median were associated with significantly shorter recurrence-free survival. PUNLMPs expressing claudin-1 over the median revealed significantly longer recurrence-free survival. High claudin-1 protein expression might help to differentiate IUP from UPs, PUNLMPs, and LG-UCCs. High claudin-4 expression may determine an unfavorable clinical course of LG-UCCs, while high claudin-1 expression in PUNLMP was associated with markedly better clinical outcome.

Novel expression of claudin-5 in glomerular podocytes

Tight junctions are the main intercellular junctions of podocytes of the renal glomerulus under nephrotic conditions. Their requisite components, claudins, still remain to be identified. We have measured the mRNA levels of claudin subtypes by quantitative real-time PCR using isolated rat glomeruli. Claudin-5 was found to be expressed most abundantly in glomeruli. Mass spectrometric analysis of membrane preparation from isolated glomeruli also confirmed only claudin-5 expression without any detection of other claudin subtypes. In situ hybridization and immunolocalization studies revealed that claudin-5 was localized mainly in glomeruli where podocytes were the only cells expressing claudin-5. Claudin-5 protein was observed on the entire surface of podocytes including apical and basal domains of the plasma membrane in the normal condition and was inclined to be concentrated on tight junctions in puromycin aminonucleoside nephrosis. Total protein levels of claudin-5 in isolated glomeruli were not significantly upregulated in the nephrosis. These findings suggest that claudin-5 is a main claudin expressed in podocytes and that the formation of tight junctions in the nephrosis may be due to local recruitment of claudin-5 rather than due to total upregulation of the claudin protein levels.

A d-Peptide Analog of the Second Extracellular Loop of Claudin-3 and -4 Leads to MisLocalized Claudin and Cellular Apoptosis in Mammary Epithelial Cells

Claudins are cell adhesion proteins thought to mediate cell-cell contacts at the tight junction. Although a major role of claudins is to control paracellular diffusion, increasing evidence suggests that they may also function in tumor progression. To examine the role of the second extracellular loop in cell adhesion, a small peptide was designed, which mimics a conserved sequence, DFYNP, within specific 'classic' claudin subtypes. Using fluorescent indicators with mammary epithelial cells, treatment with both the L- and D-forms of this peptide showed mislocalization of claudin-4 and claudin-3 and activation of caspase-8 and caspase-3, indicating apoptosis. To test specificity, peptides were made both with various end-groups and with glycine substitutions at each of the five residues. Changing end-groups did not influence the activity of the peptide. Amino acid substitutions at F147, Y148, N149, or P150, however, prevented peptide activity. A fluorescent-labeled peptide was shown to associate with the tight junction at 4 °C and cause apoptosis when the cultures were warmed to 37 °C. In conclusion, both the D- and L-forms of a small peptide that mimics a sequence in the second extracellular loop of claudins can target and disrupt claudin proteins in an epithelial monolayer and initiate apoptosis.

Invited Commentary: Role of Estrogen Receptor-α in Regulating Claudin-6 Expression in Breast Cancer Cells

Tight junctions (TJs) contribute to the paracellular barrier, which is a fence that divides plasma membranes and signal transduction, acting as a multifunctional complex in vertebrate epithelial and endothelial cells [1]. The morphological features of TJs fits well with their functions. The TJ core is a fibril-like proteinaceous structure within the lipid bilayer, the so-called TJ strands [2]. Identification and characterization of TJs-associated proteins during the last two decades has unveiled the nature of TJ strands and how they are spatially organized. The interplay between integral membrane proteins, claudins, and cytoplasmic plaque proteins, ZO-1/ZO-2, is critical for TJ formation and function [3]. Among the TJ-associated proteins, the claudin family of membrane proteins, identified in 1998 by the Tsukita group, are key molecules in the architecture and barrier function of TJs [4]. Claudins maintain cell polarity and are involved in recruiting signaling proteins. Claudins are also hypothesized to be involved in the regulation of proliferation, differentiation, and other cellular functions [5]. TJ dysfunction has been presumed as a mechanism for the loss of cell adhesion and an important step in the progression of cancer to metastasis. Because claudin expression patterns are tissue- and cell-specific, recent studies have suggested that claudins might be useful molecular markers for many different cancers [6,7]. Moreover, claudins are a potential target for novel antibody-based therapies of diverse cancers [8]. Until now, 24 members of the claudin family have been identified. The role of claudins in breast cancer is unknown and not well-explored, but recent reports have shown that claudins 3 and 4 are overexpressed in breast cancer, whereas claudins 1 and 7 are downregulated or completely absent [9-11]. Furthermore, Szasz et al. [12] indicated that the loss of claudins might contribute to breast cancer progression, and that certain claudin expression patterns might be of prognostic relevance. These results suggest that claudin subtypes may have different roles during different stages of breast cancer. Claudin-6 is expressed during the early stage of epidermal morphogenesis and is crucial to epidermal differentiation and barrier formation [13]. A recent report showed that claudin-6 is preferentially expressed in mammary epithelial cells and functions as a breast cancer tumor suppressor [14]. Additionally, Osanai et al. [15] suggested that the claudin-6 methylator phenotype might contribute to enhance tumorigenic and invasive properties of breast cancer cells. However, no information is available on the regulating mechanism of claudin-6 expression in breast cancer. In this study by Yafang et al. [16], authors demonstrated that estrogen induces claudin-6 expression through an estrogen receptor (ER) α pathway in MCF-7 cells. The data are clear, and this is the first report showing that claudin-6 expression was associated with ERα in breast cancer cells. However, first of all, authors had to indicate how claudin-6 expression is related to ERs. It was necessary to study claudin-6 expression in ER (+) and ER (-) breast cancer (prior to ERα and ERβ cells). Authors described in their previous studies that no significant association was found between claudin-6 expression and ER (+) breast cancer (even ERs). It is well accepted that the MCF-7 cell line may vary depending on in vitro culture conditions. Therefore, disparate results may occur during in vitro selection conditions. Although results from a laboratory setting may not exactly correlate with in vivo results, there is little significance to a research finding that only has significance in vitro. Because claudin-6 expression was also observed in ERβ (+) breast cancer cells, authors must show that claudin-6 is expressed in the MDA-MB-231 cancer cell line and demonstrate that the ERβ pathway is not associated with this protein expression. But this was also omitted. Thus, further investigation with a much larger cohort should be performed to confirm the relationship between claudin-6 and ERs. Claudin expression is associated with breast cancer prognosis. Down-regulation of claudin-2 is significantly associated with lymph node metastasis and high clinical stage [17]. Additionally, high levels of claudin-4 are associated with adverse outcomes in patients with breast cancer [18]. Therefore, it is also necessary to evaluate the clinicopathological parameters of breast cancers to determine whether claudin-6 could be used as a predictor of survival or prognosis.

Molecular Determinants of the Interaction between Clostridium perfringens Enterotoxin Fragments and Claudin-3

Clostridium perfringens enterotoxin (CPE) binds to the extracellular loop 2 of a subset of claudins, e.g. claudin-3. Here, the molecular mechanism of the CPE-claudin interaction was analyzed. Using peptide arrays, recombinant CPE-(116-319) bound to loop 2 peptides of mouse claudin-3, -6, -7, -9, and -14 but not of 1, 2, 4, 5, 8, 10-13, 15, 16, 18-20, and 22. Substitution peptide mapping identified the central motif (148)NPL(150)VP, supposed to represent a turn region in the loop 2, as essential for the interaction between CPE and murine claudin-3 peptides. CPE-binding assays with claudin-3 mutant-transfected HEK293 cells or lysates thereof demonstrated the involvement of Asn(148) and Leu(150) of full-length claudin-3 in the binding. CPE-(116-319) and CPE-(194-319) bound to HEK293 cells expressing claudin-3, whereas CPE-(116-319) bound to claudin-5-expressing HEK293 cells, also. This binding was inhibited by substitutions T151A and Q156E in claudin-5. In contrast, removal of the aromatic side chains in the loop 2 of claudin-3 and -5, involved in trans-interaction between claudins, increased the amount of CPE-(116-319) bound. These findings and molecular modeling indicate different molecular mechanisms of claudin-claudin trans-interaction and claudin-CPE interaction. Confocal microscopy showed that CPE-(116-319) and CPE-(194-319) bind to claudin-3 at the plasma membrane, outside cell-cell contacts. Together, these findings demonstrate that CPE binds to the hydrophobic turn and flanking polar residues in the loop 2 of claudin-3 outside tight junctions. The data can be used for the specific design of CPE-based modulators of tight junctions, to improve drug delivery, and as chemotherapeutics for tumors overexpressing claudins.