Tight Junction Strands Research Articles

Claudin-3 acts as a sealing component of the tight junction for ions of either charge and uncharged solutes

The paracellular barrier of epithelia and endothelia is established by several tight junction proteins including claudin-3. Although claudin-3 is present in many epithelia including skin, lung, kidney, and intestine and in endothelia, its function is unresolved as yet. We therefore characterized claudin-3 by stable transfection of MDCK II kidney tubule cells with human claudin-3 cDNA. Two clone systems were analyzed, exhibiting high or low claudin-2 expression, respectively. Expression of other claudins was unchanged. Ultrastructurally, tight junction strands were changed toward uninterrupted and rounded meshwork loops. Functionally, the paracellular resistance of claudin-3-transfected monolayers was strongly elevated, causing an increase in transepithelial resistance compared to vector controls. Permeabilities for mono- and divalent cations and for anions were decreased. In the high-claudin-2 system, claudin-3 reduced claudin-2-induced cation selectivity, while in the low-claudin-2 system no charge preference was observed, the latter thus reflecting the "intrinsic" action of claudin-3. Furthermore, the passage of the paracellular tracers fluorescein (332 Da) and FD-4 (4 kDa) was decreased, whereas the permeability to water was not affected. We demonstrate that claudin-3 alters the tight junction meshwork and seals the paracellular pathway against the passage of small ions of either charge and uncharged solutes. Thus, in a kidney model epithelium, claudin-3 acts as a general barrier-forming protein.

Claudin Association with CD81 Defines Hepatitis C Virus Entry

Viruses initiate infection by attaching to molecules or receptors at the cell surface. Hepatitis C virus (HCV) enters cells via a multistep process involving tetraspanin CD81, scavenger receptor class B member I, and the tight junction proteins Claudin-1 and Occludin. CD81 and scavenger receptor class B member I interact with HCV-encoded glycoproteins, suggesting an initial role in mediating virus attachment. In contrast, there are minimal data supporting Claudin-1 association with HCV particles, raising questions as to its role in the virus internalization process. In the present study we demonstrate a relationship between receptor active Claudins and their association and organization with CD81 at the plasma membrane by fluorescence resonance energy transfer and stoichiometric imaging methodologies. Mutation of residues 32 and 48 in the Claudin-1 first extracellular loop ablates CD81 association and HCV receptor activity. Furthermore, mutation of the same residues in the receptor-inactive Claudin-7 molecule enabled CD81 complex formation and virus entry, demonstrating an essential role for Claudin-CD81 complexes in HCV infection. Importantly, Claudin-1 associated with CD81 at the basolateral membrane of polarized HepG2 cells, whereas tight junction-associated pools of Claudin-1 demonstrated a minimal association with CD81. In summary, we demonstrate an essential role for Claudin-CD81 complexes in HCV infection and their localization at the basolateral surface of polarized hepatoma cells, consistent with virus entry into the liver via the sinusoidal blood and association with basal expressed forms of the receptors.

Claudin-2–deficient mice are defective in the leaky and cation-selective paracellular permeability properties of renal proximal tubules

Claudin-2 is highly expressed in tight junctions of mouse renal proximal tubules, which possess a leaky epithelium whose unique permeability properties underlie their high rate of NaCl reabsorption. To investigate the role of claudin-2 in paracellular NaCl transport in this nephron segment, we generated knockout mice lacking claudin-2 (Cldn2(-/-)). The Cldn2(-/-) mice displayed normal appearance, activity, growth, and behavior. Light microscopy revealed no gross histological abnormalities in the Cldn2(-/-) kidney. Ultrathin section and freeze-fracture replica electron microscopy revealed that, similar to those of wild types, the proximal tubules of Cldn2(-/-) mice were characterized by poorly developed tight junctions with one or two continuous tight junction strands. In contrast, studies in isolated, perfused S2 segments of proximal tubules showed that net transepithelial reabsorption of Na(+), Cl(-), and water was significantly decreased in Cldn2(-/-) mice and that there was an increase in paracellular shunt resistance without affecting the apical or basolateral membrane resistances. Moreover, deletion of claudin-2 caused a loss of cation (Na(+)) selectivity and therefore relative anion (Cl(-)) selectivity in the proximal tubule paracellular pathway. With free access to water and food, fractional Na(+) and Cl(-) excretions in Cldn2(-/-) mice were similar to those in wild types, but both were greater in Cldn2(-/-) mice after i.v. administration of 2% NaCl. We conclude that claudin-2 constitutes leaky and cation (Na(+))-selective paracellular channels within tight junctions of mouse proximal tubules.

PPARγ agonists upregulate the barrier function of tight junctions via a PKC pathway in human nasal epithelial cells

Peroxisome proliferator activated (PPAR)γ plays a critical role in the control of not only adipocyte differentiation, lipid metabolism and immunity but also the barrier functions of epithelial and endothelial cells. In the present study, to investigate effects of PPARγ agonists on the tight junctional barrier of human nasal epithelial cells (HNECs), hTERT-transfected HNECs, which highly express both PPARγ and tight junction proteins, were treated with the PPARγ agonists rosiglitazone and troglitazone. Treatment with the PPARγ agonists enhanced the barrier function of hTERT-transfected HNECs together with the upregulation of tight junction molecules claudin-1 and -4, occludin, and tricellulin at the transcriptional level. A significant increase of tight junction strands was also observed after treatment with rosiglitazone. Treatment with PPARγ agonists induced the activity of phospho-PKC in hTERT-transfected HNECs. The upregulation of the tight junction molecules in hTERT-transfected HNECs by rosiglitazone was inhibited by not only PPARγ antagonists GW9662 and T0070907, but also the panPKC inhibitor GF109203X.These findings suggest that PPARγ agonists upregulate the barrier function of tight junctions of human nasal epithelial cells via a PKC signaling pathway and could be novel drugs for protection against inhaled substances and pathogens in the airway epithelium of human nasal mucosa.

The protoplasmic or exoplasmic face association of tight junction particles cannot predict paracellular permeability or heterotypic claudin compatibility

Claudins constitute tight junction (TJ) strands and regulate paracellular permeability, which varies in the epithelial cells of various organs. Heterotypic claudin compatibility and/or the association of TJ particles to either the protoplasmic (P) or exoplasmic (E) face may be related to paracellular permeability. This study examined the relationship between the TJ morphology, heterotypic claudin compatibility and paracellular permeability using claudin-10b- or claudin-15-expressing HEK293 cells and MDCK I cells. Claudin-10b or -15 expressed in TJ-free HEK293 cells formed E-face- or P-face-associated TJ particles, respectively. The coculture of claudin-1-expressing HEK293 cells and either claudin-10b- or claudin-15-expressing HEK293 cells showed that claudin-10b and -15 were not compatible with claudin-1. The expression of claudin-10b or -15 in high-resistance MDCK I cells did not alter the expression of endogenous claudins except for claudin-3 and dramatically reduced transepithelial electrical resistance by increasing the permeability of Na + but it did not change that of Cl −. The expression of claudin-10b or -15 in MDCK I cells either decreased or increased the flux of 4 kDa dextran, respectively. The coculture of MDCK I cells and either claudin-10b- or claudin-15-expressing MDCK I cells showed claudin-10b to be partly compatible, while claudin-15 was incompatible with the endogenous claudins in MDCK I cells. These results indicate that the TJ morphology cannot predict the properties of either paracellular permeability or heterotypic claudin compatibility.

NaCl flux between apical and basolateral side recruits claudin-1 to tight junction strands and regulates paracellular transport

In multicellular organisms, epithelia separate and divide the internal environment maintaining appropriate conditions in each compartment. To maintain homeostasis in these compartments, claudins, major cell adhesion molecules in tight junctions (TJs), regulate movements of several substances through the paracellular pathway (barrier function). In this study, we investigated effects of the flux of several substances between apical and basolateral side on paracellular transport and TJ protein localization. NaCl flux from apical to basolateral side increased paracellular conductance (Gp) and recruited claudin-1 from lateral cell membrane to the apical end with the colocalization with occludin, one of the TJ proteins concentrated at TJ strands. Oppositely-directed flux of sucrose against NaCl flux inhibited these reactions and same directional flux of sucrose with NaCl enhanced the increase of Gp, whereas 10-kDa dextran inhibited these reactions regardless of the side of administration. Our present findings indicated that TJ protein localization and barrier function are regulated depending on the environmental differences between apical and basolateral side.

The Structure of Tight Junctions in Mouse Submandibular Gland

Salivary gland cells are joined by junctional complexes consisting of a tight junction (TJ), zonula adherens and one or more desmosomes. TJs regulate paracellular permeability, maintain separate apical and basolateral membrane domains, and serve as signaling centers. We examined TJs of mouse submandibular glands (SMG) in thin sections and freeze-fracture replicas. TJs between acinar cells and between intercalated duct cells had 2-6 parallel strands on the protoplasmic fracture face, with occasional branches, interconnections and free ends, and corresponding grooves on the extracellular face. Granular duct cell TJs had 2-30 strands, a depth of <or=0.5 microm, and occasional loops extending further basally. Where 3 or 4 cells met, the TJs extended basally <or=1 microm and consisted of 2 parallel boundary strands into which the apical strands inserted. Quantitative analyses showed significant differences in TJ complexity, measured by fractal geometry, and strand number of acinar compared to granular duct cells, and a greater number of strands in male compared to female granular ducts. Pilocarpine stimulation increased TJ strand number in female acinar cells, and increased complexity of male granular duct cell TJs. As the salivary gland water channel aquaporin 5 (AQP5) has been proposed to functionally interact with TJs to regulate salivary fluid composition, we also studied glands from AQP5 knock-out mice. In males lacking AQP5, granular duct TJs were more complex than those of wild-type mice, and exhibited more strands following pilocarpine stimulation. The results demonstrate specific gender, cell type and genetic differences in TJ structure and response to stimulation.

Arcobacter butzleriInduces Barrier Dysfunction in Intestinal HT‐29/B6 Cells

Arcobacter butzleri causes watery diarrhea and bacteremia. Although, recently, more cases of diarrhea have been caused by Arcobacter species, very little is known about its pathogenesis, the identification of which is the aim of this study. Human HT-29/B6 colonic epithelial monolayers were apically inoculated with A. butzleri. Transepithelial resistance and macromolecule fluxes were measured in Ussing chambers. Tight junction protein expression was analyzed by Western blotting, and subcellular distribution was analyzed by confocal laser-scanning microscopy. Infection of HT-29/B6 caused a decrease in transepithelial resistance to 30% and an increase in paracellular permeability to fluorescein (10.8+/-3.5 10(-6) cm/s vs. 1.8+/-0.6 10(-6) cm/s in control; P<.05) and dextran-4 kDa (0.036+/-0.005 10(-6) cm/s vs. 0.015+/-0.002 10(-6) cm/s in control; P<.01). This effect was time and dose dependent and was also caused by bacterial lysates showing heat and proteinase-K sensitivity. As structural correlate, expression of the tight junctional proteins claudin-1, -5, and -8 was reduced, and claudin-1 and -8 were redistributed off the tight junctional strands forming intracellular aggregates. Furthermore, A. butzleri induced epithelial apoptosis (3-fold). A. butzleri induces epithelial barrier dysfunction by changes in tight junction proteins and induction of epithelial apoptosis, which are mechanisms that are consistent with a leak flux type of diarrhea in A. butzleri infection.

A Claudin-9–Based Ion Permeability Barrier Is Essential for Hearing

Hereditary hearing loss is one of the most common birth defects, yet the majority of genes required for audition is thought to remain unidentified. Ethylnitrosourea (ENU)–mutagenesis has been a valuable approach for generating new animal models of deafness and discovering previously unrecognized gene functions. Here we report on the characterization of a new ENU–induced mouse mutant (nmf329) that exhibits recessively inherited deafness. We found a widespread loss of sensory hair cells in the hearing organs of nmf329 mice after the second week of life. Positional cloning revealed that the nmf329 strain carries a missense mutation in the claudin-9 gene, which encodes a tight junction protein with unknown biological function. In an epithelial cell line, heterologous expression of wild-type claudin-9 reduced the paracellular permeability to Na+ and K+, and the nmf329 mutation eliminated this ion barrier function without affecting the plasma membrane localization of claudin-9. In the nmf329 mouse line, the perilymphatic K+ concentration was found to be elevated, suggesting that the cochlear tight junctions were dysfunctional. Furthermore, the hair-cell loss in the claudin-9–defective cochlea was rescued in vitro when the explanted hearing organs were cultured in a low-K+ milieu and in vivo when the endocochlear K+-driving force was diminished by deletion of the pou3f4 gene. Overall, our data indicate that claudin-9 is required for the preservation of sensory cells in the hearing organ because claudin-9–defective tight junctions fail to shield the basolateral side of hair cells from the K+-rich endolymph. In the tight-junction complexes of hair cells, claudin-9 is localized specifically to a subdomain that is underneath more apical tight-junction strands formed by other claudins. Thus, the analysis of claudin-9 mutant mice suggests that even the deeper (subapical) tight-junction strands have biologically important ion barrier function.

Molecular Basis of the Core Structure of Tight Junctions

The morphological feature of tight junctions (TJs) fits well with their functions. The core of TJs is a fibril-like proteinaceous structure within the lipid bilayer, the so-called TJ strands. TJ strands in apposing plasma membranes associate with each other to eliminate the intercellular space. A network of paired TJ strands generates a continuous belt that circumscribes each cell to establish the diffusion barrier to the solutes in the paracellular pathway throughout the cellular sheet. Identification and characterization of TJ-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.

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.

A protein diffusion model of the sealing effect.

Water transport across the arterial endothelium is believed primarily to occur through breaks in the tight junction strands at the cell periphery between neighboring cells. Additional proteins arriving at the tight junction can close these breaks, thereby attenuating this water flux. Motivated by evidence that the diffusion of presynthesized protein from the interior of the cell to and incorporation into the cell border is the mechanism of endothelial tight junctional sealing, we develop a diffusion-limited mathematical model of intercellular gap sealing. A single endothelial cell is represented as a thin, axisymmetric disk, initially containing a uniform distribution of junctional protein that does not interact with the apical or basal cell surfaces. Upon application of a transmural pressure gradient, water flows through the junctional cleft, and tight junction remodeling begins. We assume that proteins at the junction are instantaneously incorporated into its strand, dropping the free protein concentration at the cell periphery to zero. This sets the diffusion of intracellular proteins toward the junction in motion. The solution of this one-dimensional initial value problem provides excellent fits to current and previously published experimental data over a wide variety of conditions. It yields three physically meaningful parameters for each fit, including a protein diffusivity in the cytoplasm that varies little within experimental treatments. Statistical variation of these parameters allows rational comparison of experimental runs and identification of outlier runs.

Roles of ZO‐1 and ZO‐2 in Establishment of the Belt‐like Adherens and Tight Junctions with Paracellular Permselective Barrier Function

Tight junctions (TJs) create the primary permselective barrier to diffusion of solutes and ions through the paracellular pathway. The molecular architecture of TJs has gradually been unraveled in recent years, providing the basis for "barriology" (defined by Shoichiro Tsukita as the science of the barrier in multicellular organisms). Claudins are now considered to be the essential basic components of TJ strands, with which other integral membrane proteins, such as occludin, tricellulin, JAMs, and CAR, are associated. Peripherally associated scaffolding proteins are required for the organization of the integral membrane proteins. Among these, ZO-1, -2, and -3 have attracted a great deal of attention as TJ organizers, since ZO-1 (and in some cases, also ZO-2/3) was reported to be directly associated with claudins, occludin, and JAMs, as well as with AF-6/afadin and alpha-catenin. Here we summarize recent studies on ZO-1/2/3-deficiency in mice and cells, which have provided clear and important information regarding the functions of ZO-1/2/3 in vivo. In addition to the respective suppression of ZO-1/2/3 expression, simultaneous suppression of all three proteins has revealed the essential and nonessential in vivo roles of ZO-1/2 and ZO-3, respectively. ZO-3 shows an epithelial-specific TJ localization in a ZO-1/2-dependent fashion. ZO-1 and ZO-2 play pivotal roles in the final establishment of the belt-like adherens junctions (zonula adherens), followed by the formation of the belt-like TJs (zonula occludens) with paracellular barrier function, thereby providing the general basis for selective paracellular permeability in epithelial and endothelial cells.

Epithelial Tight Junctions in Intestinal Inflammation

The epithelium in inflamed intestinal segments of patients with Crohn's disease is characterized by a reduction of tight junction strands, strand breaks, and alterations of tight junction protein content and composition. In ulcerative colitis, epithelial leaks appear early due to micro-erosions resulting from upregulated epithelial apoptosis and in addition to a prominent increase of claudin-2. Th1-cytokine effects by interferon-gamma in combination with TNFalpha are important for epithelial damage in Crohn's disease, while interleukin-13 (IL-13) is the key effector cytokine in ulcerative colitis stimulating apoptosis and upregulation of claudin-2 expression. Focal lesions caused by apoptotic epithelial cells contribute to barrier disturbance in IBD by their own conductivity and by confluence toward apoptotic foci or erosions. Another type of intestinal barrier defect can arise from alpha-hemolysin harboring E. coli strains among the physiological flora, which can gain pathologic relevance in combination with proinflammatory cytokines under inflammatory conditions. On the other hand, intestinal barrier impairment can also result from transcellular antigen translocation via an initial endocytotic uptake into early endosomes, and this is intensified by proinflammatory cytokines as interferon-gamma and may thus play a relevant role in the onset of IBD. Taken together, barrier defects contribute to diarrhea by a leak flux mechanism (e.g., in IBD) and can cause mucosal inflammation by luminal antigen uptake. Immune regulation of epithelial functions by cytokines may cause barrier dysfunction not only by tight junction impairments but also by apoptotic leaks, transcytotic mechanisms, and mucosal gross lesions.

Tight Junction Proteins as Channel Formers and Barrier Builders

Tight junctions form the paracellular barrier for ions and uncharged solutes not only in "tight" but also in "leaky" epithelia. In the premolecular era of tight junction research, this was believed to be achieved in a perfect or less perfect way, depending mainly on the amount of horizontally oriented tight junction strands. During the past decade it emerged that tight junction molecules, such as claudin-1 and many others, strengthen the barrier, while a few claudins, such as claudin-2 or -10, weaken it. This report focuses on three claudins: one channel former and two barrier builders. Claudin-2 represents the prototype of a paracellular, channel-forming, tight junction protein responsible for specific transfer of solutes across the epithelium without entering the cells. This channel is selective for small cations but nearly impermeable to anions and uncharged solutes of any size. In contrast, claudin-5, a tight junction protein typical for all endothelia but also found in some epithelia, was characterized as a potent barrier builder. Claudin-8, another barrier builder, was demonstrated to be regulated by Na(+) uptake in surface epithelial cells of human colon. Here, aldosterone enhanced Na(+) absorption by dual action: transcellularly by inducing the epithelial sodium channel and paracellularly by preventing back leakage of absorbed Na(+) by upregulating claudin-8.

The E3 ubiquitin ligase LNX1p80 promotes the removal of claudins from tight junctions in MDCK cells

The structural continuity of tight junctions (TJs) is consistently maintained even when epithelial cells divide and move within the cellular sheet. This process is associated with dynamic remodeling of TJs by coordinated internalization and generation of claudin-based TJ strands, but the molecular mechanism behind the regulated turnover of TJs remains largely unknown. In this study, we identified the p80 isoform of the E3 ubiquitin ligase ligand of Numb-protein X1 (LNX1p80) as a protein binding to claudin-1. Interestingly, the concentration of claudins in TJs was remarkably reduced when LNX1p80 was overexpressed in MDCK cells, and there was a reduction not only in the number of TJ strands but also in the amount of detergent-insoluble claudins. We also found that LNX1p80 promoted polyubiquitylation of claudins. This ubiquitylation is dependent on its RING-finger domain and is not mediated by Lys48 of ubiquitin, which is used for protein degradation by the proteasome. Furthermore, LNX1p80 was often colocalized with claudins in vesicular structures containing markers for late endosomes and lysosomes. These findings suggest that LNX1p80 is involved in the ubiquitylation, endocytosis and lysosomal degradation of claudins, and that the turnover of TJs is regulated by ubiquitylation.

Freeze-fracture electron microscopic study of tight junction strands in HEK293 cells and MDCK II cells expressing claudin-1 mutants in the second extracellular loop

Claudins constitute tight junction (TJ) strands. In order to examine the function of the second extracellular loop (ECL2), we constructed 1CLDeltaFY and 1CLDeltaPL in which highly conserved amino acids, FY or PL, in the ECL2 of mouse claudin-1 were deleted. They were then tagged with either EGFP at the NH(2)-terminus (EGFP1CLDeltaFY and EGFP1CLDeltaPL) or the myc-epitope at the COOH-terminus (1CLDeltaFYmyc and 1CLDeltaPLmyc). The expression of EGFP1CLDeltaFY and EGFP1CLDeltaPL in TJ-free HEK293 cells formed TJ strands resembling those formed by wild-type claudin-1. The expression of 1CLDeltaPLmyc in TJ-bearing MDCK II cells induced aberrant TJ strands in the lateral plasma membranes whose intramembranous particles were almost equally distributed in the P- and E-face. In contrast, 1CLDeltaFYmyc formed aggregates of short continuous strands which were frequently associated with vesicle-like structures. Coculture experiments with MDCK II cells showed that 1CLDeltaPLmyc was localized at heterotypic cell-cell junctions but 1CLDeltaFYmyc was not. These results suggest that changes in the TJ morphology due to the expression of either 1CLDeltaFYmyc or 1CLDeltaPLmyc may be caused by some factors specific to epithelial MDCK II cells including endogenous claudins.

Regulation of tight junction permeability by sodium caprate in human keratinocytes and reconstructed epidermis

Tight junctions (TJs) restrict paracellular flux of water and solutes in epithelia and endothelia. In epidermis, the physiological role of TJs is not fully understood. In this study, sodium caprate (C10), which dilates intestinal TJs, was applied to cultured human epidermal keratinocytes and reconstructed human epidermis to investigate the effects of C10 on epidermal TJs. C10 treatment decreased transepithelial electrical resistance and increased paracellular permeability, although Western blots showed that the expression of TJ-related transmembrane proteins was not decreased. The effects of C10 were reversible. Immunofluorescence microscopy and immuno-replica electron microscopy showed that the localization of TJ strands were disintegrated, concomitant with the dispersion and/or disappearance of TJ-related molecules from the cell surface. These findings suggest that C10 impairs barrier function by physically disrupting TJ conformation in the epidermis. Furthermore, these results also show that proper localization of the molecules on the cellular membrane is important for TJ barrier function.

Tight Junction Transmembrane Protein Claudin Subtype Expression and Distribution in Human Corneal and Conjunctival Epithelium

The combination of the tight junction transmembrane protein claudin subtypes is one of the most important determinants of variations in the tightness of individual paired tight junction strands. The barrier function of corneal epithelium is much stronger than that of conjunctival epithelium. In this study, the expression and cellular distribution of claudin species in in vivo human corneal and conjunctival epithelium were investigated. Reverse transcription-polymerase chain reaction was used to reveal the claudin mRNA. Immunohistochemistry was used to determine the tissue distribution of tight junction-related proteins and MUC5AC. Transcripts for claudin-1, -2, -3, -4, -7, -9, and -14 were identified in human corneal epithelium. Transcripts for claudin-1, -2, -4, -7, -9, -10, and -14 were identified in human conjunctival epithelium. By immunohistochemistry, claudin-1, -4, and -7 were found to be localized at the membrane of human corneal and conjunctival epithelial cells. In human conjunctival epithelium, claudin-10 staining was observed at several, but not all, apical epithelial cell-to-goblet cell junctions. Claudin-1, -4, and -7 are expressed in corneal and conjunctival epithelia. Claudin-10 is prominent at several junctions between apical epithelial cells and goblet cells in conjunctival epithelium. Except for claudin-10 expression in conjunctival epithelium, the claudin subtype expressions of corneal and conjunctival epithelia are similar. Therefore, there must be a difference between these two epithelial types with regard to the specific ratio of claudin subtypes expressed or their phosphorylation status. The distribution of goblet cells in conjunctival epithelium also influences the difference in barrier function.

Knockdown of tight junction protein claudin-2 prevents bile canalicular formation in WIF-B9 cells

The polarization of hepatocytes involves formation of functionally distinct sinusoidal (basolateral) and bile canalicular (apical) plasma membrane domains that are separated by tight junctions. Although various molecular mechanisms and signaling cascades including polarity complex proteins may contribute to bile canalicular formation in hepatocytes, the role of tight junction proteins in bile canalicular formation remains unclear. To investigate the role of the integral tight junction protein claudin-2 in bile canalicular formation, we depleted claudin-2 expression by siRNA in the polarized hepatic cell line WIF-B9 after treatment with or without phenobarbital. When WIF-B9 cells were treated with phenobarbital, claudin-2 expression and tight junction strands were markedly increased together with induction of canalicular formation with a biliary secretion function. Knockdown of claudin-2 prevented bile canalicular formation after treatment with or without phenobarbital. Furthermore, knockdown of claudin-2 caused a change from a hepatic polarized phenotype to a simple polarized phenotype, together with upregulation of pLKB1, pMAPK, pAkt and pp38 MAPK, but not pMLC, PTEN or cdc42, and an increase of intracellular vacuoles, which were present before bile canalicular formation. These results suggest that claudin-2 may affect not only the bile canalicular seal but also bile canalicular formation.