Epidermal growth factor receptor activation differentially regulates claudin expression and enhances transepithelial resistance in Madin-Darby canine kidney cells

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

The degradation of airway tight junction protein under acidic conditions is probably mediated by transient receptor potential vanilloid 1 receptor

Acidic airway microenvironment is one of the representative pathophysiological features of chronic inflammatory respiratory diseases. Epithelial barrier function is maintained by TJs (tight junctions), which act as the first physical barrier against the inhaled substances and pathogens of airway. As previous studies described, acid stress caused impaired epithelial barriers and led the hyperpermeability of epithelium. However, the specific mechanism is still unclear. We have showed previously the existence of TRPV (transient receptor potential vanilloid) 1 channel in airway epithelium, as well as its activation by acidic stress in 16HBE cells. In this study, we explored the acidic stress on airway barrier function and TJ proteins in vitro with 16HBE cell lines. Airway epithelial barrier function was determined by measuring by TER (trans-epithelial electrical resistance). TJ-related protein [claudin-1, claudin-3, claudin-4, claudin-5, claudin-7 and ZO-1 (zonula occluden 1)] expression was examined by western blotting of insoluble fractions of cell extraction. The localization of TJ proteins were visualized by immunofluorescent staining. Interestingly, stimulation by pH 6.0 for 8 h slightly increased the epithelial resistance in 16HBE cells insignificantly. However, higher concentration of hydrochloric acid (lower than pH 5.0) did reduce the airway epithelial TER of 16HBE cells. The decline of epithelial barrier function induced by acidic stress exhibited a TRPV1-[Ca²⁺]_i-dependent pathway. Of the TJ proteins, claudin-3 and claudin-4 seemed to be sensitive to acidic stress. The degradation of claudin-3 and claudin-4 induced by acidic stress could be attenuated by the specific TRPV1 blocker or intracellular Ca²⁺ chelator BAPTA/AM [1,2-bis-(o-aminophenoxy)ethane-N,N,N'',N''-tetra-acetic acid tetrakis(acetoxymethyl ester)].     

Androgen initiates Sertoli cell tight junction formation in the hypogonadal (hpg) mouse

Sertoli cell tight junctions (TJs) form at puberty as a major component of the blood-testis barrier (BTB), which is essential for spermatogenesis. This study characterized the hormonal induction of functional Sertoli cell TJ formation in vivo using the gonadotropin-deficient hypogonadal (hpg) mouse that displays prepubertal spermatogenic arrest. Androgen actions were determined in hpg mice treated for 2 or 10 days with dihydrotestosterone (DHT). Follicle-stimulating hormone (FSH) actions were studied in hpg mice expressing transgenic human FSH (hpg+tgFSH) with or without DHT treatment. TJ formation was examined by mRNA expression and immunolocalization of TJ proteins claudin-3 and claudin-11, and barrier functionality was examined by biotin tracer permeability. Immunolocalization of claudin-3 and claudin-11 was extensive at wild-type (wt) Sertoli cell TJs, which functionally excluded permeability tracer. In contrast, seminiferous tubules of hpg testes lacked claudin-3, but claudin-11 protein was present in adluminal regions of Sertoli cells. Biotin tracer permeated throughout these tubules, demonstrating dysfunctional TJs. In hpg+tgFSH testes, claudin-3 was generally absent, but claudin-11 had redistributed basally toward the TJs, where function was variable. In hpg testes, DHT treatment stimulated the redistribution of claudin-11 protein toward the basal region of Sertoli cells by Day 2, increased Cldn3 and Cldn11 mRNA expression, then induced the formation of functional TJs containing both proteins by Day 10. In hpg+tgFSH testes, TJ protein redistribution was accelerated and functional TJs formed by Day 2 of DHT treatment. We conclude that androgen stimulates initial Sertoli cell TJ formation and function in mice, whereas FSH activity is insufficient alone, but augments androgen-induced TJ function.     

Enteropathogenic Escherichia coli infection leads to appearance of aberrant tight junctions strands in the lateral membrane of intestinal epithelial cells

Infection of intestinal epithelial cells with enteropathogenic Escherichia coli (EPEC) disrupts tight junction (TJ) architecture and barrier function. The aim of this study was to determine the impact of EPEC on TJ protein interactions and localization. Human intestinal epithelial cells (T84) were infected for 1, 3 or 6 h with EPEC. To probe the TJ protein-protein interactions, co-immunoprecipitations were performed. The associations between ZO-1, occludin and claudin-1 progressively decreased after infection. Corresponding morphological changes were analysed by immunofluorescence confocal microscopy. Tight junction proteins progressively lost their apically restricted localization. Freeze-fracture electron microscopy revealed the appearance of aberrant strands throughout the lateral membrane that contained claudin-1 and occludin as determined by immunogold labelling. These structural alterations were accompanied by a loss of barrier function. Mutation of the gene encoding EspF, important in the disruption of TJs by EPEC, prevented the disruption of TJs. Tight junction structure normalized following eradication of EPEC with gentamicin and overnight recovery. This is the first demonstration that a microbial pathogen can cause aberrant TJ strands in the lateral membrane of host cells. We speculate that the disruption of integral and cytoplasmic TJ protein interactions following EPEC infection allows TJ strands to form or diffuse into the lateral plasma membrane.     

Phosphorylation of claudin-4 by PKCepsilon regulates tight junction barrier function in ovarian cancer cells

Claudin proteins belong to a large family of transmembrane proteins essential to the formation and maintenance of tight junctions (TJs). In ovarian cancer, TJ protein claudin-4 is frequently overexpressed and may have roles in survival and invasion, but the molecular mechanisms underlying its regulation are poorly understood. In this report, we show that claudin-4 can be phosphorylated by protein kinase C (PKC) at Thr189 and Ser194 in ovarian cancer cells and overexpression of a claudin-4 mutant protein mimicking the phosphorylated state results in the disruption of the barrier function. Furthermore, upon phorbol ester-mediated PKC activation of OVCA433 cells, TJ strength is decreased and claudin-4 localization is altered. Analyses using PKC inhibitors and siRNA suggest that PKCepsilon, an isoform typically expressed in ovarian cancer cells, may be important in the TPA-mediated claudin-4 phosphorylation and weakening of the TJs. Furthermore, immunofluorescence studies showed that claudin-4 and PKCepsilon are co-localized at the TJs in these cells. The modulation of claudin-4 activity by PKCepsilon may not only provide a mechanism for disrupting TJ function in ovarian cancer, but may also be important in the regulation of TJ function in normal epithelial cells.     

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.     

Clostridium perfringens enterotoxin fragment removes specific claudins from tight junction strands: Evidence for direct involvement of claudins in tight junction barrier.

Claudins, comprising a multigene family, constitute tight junction (TJ) strands. Clostridium perfringens enterotoxin (CPE), a single approximately 35-kD polypeptide, was reported to specifically bind to claudin-3/RVP1 and claudin-4/CPE-R at its COOH-terminal half. We examined the effects of the COOH-terminal half fragment of CPE (C-CPE) on TJs in L transfectants expressing claudin-1 to -4 (C1L to C4L, respectively), and in MDCK I cells expressing claudin-1 and -4. C-CPE bound to claudin-3 and -4 with high affinity, but not to claudin-1 or -2. In the presence of C-CPE, reconstituted TJ strands in C3L cells gradually disintegrated and disappeared from their cell surface. In MDCK I cells incubated with C-CPE, claudin-4 was selectively removed from TJs with its concomitant degradation. At 4 h after incubation with C-CPE, TJ strands were disintegrated, and the number of TJ strands and the complexity of their network were markedly decreased. In good agreement with the time course of these morphological changes, the TJ barrier (TER and paracellular flux) of MDCK I cells was downregulated by C-CPE in a dose-dependent manner. These findings provided evidence for the direct involvement of claudins in the barrier functions of TJs.     

Conversion of zonulae occludentes from tight to leaky strand type by introducing claudin-2 into Madin-Darby canine kidney I cells

There are two strains of MDCK cells, MDCK I and II. MDCK I cells show much higher transepithelial electric resistance (TER) than MDCK II cells, although they bear similar numbers of tight junction (TJ) strands. We examined the expression pattern of claudins, the major components of TJ strands, in these cells: claudin-1 and -4 were expressed both in MDCK I and II cells, whereas the expression of claudin-2 was restricted to MDCK II cells. The dog claudin-2 cDNA was then introduced into MDCK I cells to mimic the claudin expression pattern of MDCK II cells. Interestingly, the TER values of MDCK I clones stably expressing claudin-2 (dCL2-MDCK I) fell to the levels of MDCK II cells (>20-fold decrease). In contrast, when dog claudin-3 was introduced into MDCK I cells, no change was detected in their TER. Similar results were obtained in mouse epithelial cells, Eph4. Morphometric analyses identified no significant differences in the density of TJs or in the number of TJ strands between dCL2-MDCK I and control MDCK I cells. These findings indicated that the addition of claudin-2 markedly decreased the tightness of individual claudin-1/4-based TJ strands, leading to the speculation that the combination and mixing ratios of claudin species determine the barrier properties of individual TJ strands.     

Multi-PDZ domain protein 1 (MUPP1) is concentrated at tight junctions through its possible interaction with claudin-1 and junctional adhesion molecule.

Claudins, most of which end in valine at their COOH termini, constitute tight junction (TJ) strands, suggesting that TJ strands strongly attract PDZ-containing proteins. Indeed, ZO-1, -2, and -3, each of which contains three PDZ domains, were shown to directly bind to claudins. Using the yeast two-hybrid system, we identified ZO-1 and MUPP1 (multi-PDZ domain protein 1) as binding partners for the COOH terminus of claudin-1. MUPP1 has been identified as a protein that contains 13 PDZ domains, but it has not been well characterized. In vitro binding assays with recombinant MUPP1 confirmed the interaction between MUPP1 and claudin-1 and identified PDZ10 as the responsible domain for this interaction. A polyclonal antibody specific for MUPP1 was then generated. Immunofluorescence confocal microscopy as well as immunoelectron microscopy with this antibody revealed that in polarized epithelial cells MUPP1 was exclusively concentrated at TJs. Furthermore, in vitro binding and transfection experiments showed that junctional adhesion molecule, another TJ adhesion molecule, also bound to the PDZ9 domain of MUPP1. These findings suggested that MUPP1 is concentrated at TJs in epithelial cells through its binding to claudin and junctional adhesion molecule and that it may function as a multivalent scaffold protein that recruits various proteins to TJs.     

Phosphorylation of claudin-3 at threonine 192 by cAMP-dependent protein kinase regulates tight junction barrier function in ovarian cancer cells

Claudins are integral membrane proteins essential in the formation and function of tight junctions (TJs). Disruption of TJs, which have essential roles in cell permeability and polarity, is thought to contribute to epithelial tumorigenesis. Claudin-3 and -4 are frequently overexpressed in ovarian cancer, but the molecular pathways involved in the regulation of these proteins are unclear. Interestingly, several studies have demonstrated a role for phosphorylation in the regulation of TJ complexes, although evidence for claudin phosphorylation is scarce. Here, we showed that claudin-3 and -4 can be phosphorylated in ovarian cancer cells. In vitro phosphorylation assays using glutathione S-transferase fusion constructs demonstrated that the C terminus of claudin-3 is an excellent substrate for cAMP-dependent protein kinase (PKA). Using site-directed mutagenesis, we identified a PKA phosphorylation site at amino acid 192 in the C terminus of claudin-3. Overexpression of the protein containing a T192D mutation, mimicking the phosphorylated state, resulted in a decrease in TJ strength in ovarian cancer cell line OVCA433. Our results suggest that claudin-3 phosphorylation by PKA, a kinase frequently activated in ovarian cancer, may provide a mechanism for the disruption of TJs in this cancer. In addition, our findings may have general implications for the regulation of TJs in normal epithelial cells.     

Role of claudin species-specific dynamics in reconstitution and remodeling of the zonula occludens

Tight-junction strands, which are organized into the beltlike cell-cell adhesive structure called the zonula occludens (TJ), create the paracellular permselective barrier in epithelial cells. The TJ is constructed on the basis of the zonula adherens (AJ) by polymerized claudins in a process mediated by ZO-1/2, but whether the 24 individual claudin family members play different roles at the TJ is unclear. Here we established a cell system for examining the polymerization of individual claudins in the presence of ZO-1/2 using an epithelial-like cell line, SF7, which lacked endogenous TJs and expressed no claudin but claudin-12 in immunofluorescence and real-time PCR assays. In stable SF7-derived lines, exogenous claudin-7, -14, or -19, but no other claudins, individually reconstituted TJs, each with a distinct TJ-strand pattern, as revealed by freeze-fracture analyses. Fluorescence recovery after photobleaching (FRAP) analyses of the claudin dynamics in these and other epithelial cells suggested that slow FRAP-recovery dynamics of claudins play a critical role in regulating their polymerization around AJs, which are loosely coupled with ZO-1/2, to form TJs. Furthermore, the distinct claudin stabilities in different cell types may help to understand how TJs regulate paracellular permeability by altering the paracellular flux and the paracellular ion permeability.     

Salmonella enterica serovar Typhimurium regulates intercellular junction proteins and facilitates transepithelial neutrophil and bacterial passage

The establishment of tight junctions (TJ) between columnar epithelial cells defines the functional barrier, which enteroinvasive pathogens have to overcome. Salmonella enterica serovar Typhimurium (S. typhimurium) directly invades intestinal epithelial cells but it is not well understood how the pathogen is able to overcome the intestinal barrier and gains access to the circulation. Therefore, we sought to determine whether infection with S. typhimurium could regulate the molecular composition of the TJ and, if so, whether these modifications would influence bacterial translocation and polymorphonuclear leukocyte (PMN) movement across model intestinal epithelium. We found that infection of a model intestinal epithelium with S. typhimurium over 2 h resulted in an approximately 80% loss of transepithelial electrical resistance. Western blot analysis of epithelial cell lysates demonstrated that S. typhimurium regulated the distribution of the TJ complex proteins claudin-1, zonula occludens (ZO)-2, and E-cadherin in Triton X-100-soluble and insoluble fractions. In addition, S. typhimurium was specifically able to dephosphorylate occludin and degrade ZO-1. This TJ alteration in the epithelial monolayer resulted in 10-fold increase in bacterial translocation and a 75% increase in N-formylmethionin-leucyl-phenyalanine-induced PMN transepithelial migration. Our data demonstrate that infection with S. typhimurium is associated with the rapid targeting of the tight junctional complex and loss of barrier function. This results in enhanced bacterial translocation and initiation of PMN migration across the intestinal barrier. Therefore, the ability to regulate the molecular composition of TJs facilitates the pathogenicity of S. typhimurium by aiding its uptake and distribution within the host.     

Endothelial claudin: claudin-5/TMVCF constitutes tight junction strands in endothelial cells.

Tight junctions (TJs) in endothelial cells are thought to determine vascular permeability. Recently, claudin-1 to -15 were identified as major components of TJ strands. Among these, claudin-5 (also called transmembrane protein deleted in velo-cardio-facial syndrome [TMVCF]) was expressed ubiquitously, even in organs lacking epithelial tissues, suggesting the possible involvement of this claudin species in endothelial TJs. We then obtained a claudin-6-specific polyclonal antibody and a polyclonal antibody that recognized both claudin-5/TMVCF and claudin-6. In the brain and lung, immunofluorescence microscopy with these polyclonal antibodies showed that claudin-5/TMVCF was exclusively concentrated at cell-cell borders of endothelial cells of all segments of blood vessels, but not at those of epithelial cells. Immunoreplica electron microscopy revealed that claudin-5/TMVCF was a component of TJ strands. In contrast, in the kidney, the claudin-5/TMVCF signal was restricted to endothelial cells of arteries, but was undetectable in those of veins and capillaries. In addition, in all other tissues we examined, claudin-5/TMVCF was specifically detected in endothelial cells of some segments of blood vessels, but not in epithelial cells. Furthermore, when claudin-5/TMVCF cDNA was introduced into mouse L fibroblasts, TJ strands were reconstituted that resembled those in endothelial cells in vivo, i.e., the extracellular face-associated TJs. These findings indicated that claudin-5/TMVCF is an endothelial cell-specific component of TJ strands.     

Tight junctions in Schwann cells of peripheral myelinated axons: a lesson from claudin-19-deficient mice

Tight junction (TJ)-like structures have been reported in Schwann cells, but their molecular composition and physiological function remain elusive. We found that claudin-19, a novel member of the claudin family (TJ adhesion molecules in epithelia), constituted these structures. Claudin-19-deficient mice were generated, and they exhibited behavioral abnormalities that could be attributed to peripheral nervous system deficits. Electrophysiological analyses showed that the claudin-19 deficiency affected the nerve conduction of peripheral myelinated fibers. Interestingly, the overall morphology of Schwann cells lacking claudin-19 expression appeared to be normal not only in the internodal region but also at the node of Ranvier, except that TJs completely disappeared, at least from the outer/inner mesaxons. These findings have indicated that, similar to epithelial cells, Schwann cells also bear claudin-based TJs, and they have also suggested that these TJs are not involved in the polarized morphogenesis but are involved in the electrophysiological "sealing" function of Schwann cells.     

Regulation of airway tight junctions by proinflammatory cytokines

Epithelial tight junctions (TJs) provide an important route for passive electrolyte transport across airway epithelium and provide a barrier to the migration of toxic materials from the lumen to the interstitium. The possibility that TJ function may be perturbed by airway inflammation originated from studies reporting (1) increased levels of the proinflammatory cytokines interleukin-8 (IL-8), tumor necrosis factor alpha (TNF-alpha), interferon gamma (IFN-gamma), and IL-1beta in airway epithelia and secretions from cystic fibrosis (CF) patients and (2) abnormal TJ strands of CF airways as revealed by freeze-fracture electron microscopy. We measured the effects of cytokine exposure of CF and non-CF well-differentiated primary human airway epithelial cells on TJ properties, including transepithelial resistance, paracellular permeability to hydrophilic solutes, and the TJ proteins occludin, claudin-1, claudin-4, junctional adhesion molecule, and ZO-1. We found that whereas IL-1beta treatment led to alterations in TJ ion selectivity, combined treatment of TNF-alpha and IFN-gamma induced profound effects on TJ barrier function, which could be blocked by inhibitors of protein kinase C. CF bronchi in vivo exhibited the same pattern of expression of TJ-associated proteins as cultures exposed in vitro to prolonged exposure to TNF-alpha and IFN-gamma. These data indicate that the TJ of airway epithelia exposed to chronic inflammation may exhibit parallel changes in the barrier function to both solutes and ions.     

RhoA, Rac1, and Cdc42 exert distinct effects on epithelial barrier via selective structural and biochemical modulation of junctional proteins and F-actin

Epithelial intercellular junctions regulate cell-cell contact and mucosal barrier function. Both tight junctions (TJs) and adherens junctions (AJs) are regulated in part by their affiliation with the F-actin cytoskeleton. The cytoskeleton in turn is influenced by Rho family small GTPases such as RhoA, Rac1, and Cdc42, all of which constitute eukaryotic targets for several pathogenic organisms. With a tetracycline-repressible system to achieve regulated expression in Madin-Darby canine kidney (MDCK) epithelial cells, we used dominant-negative (DN) and constitutively active (CA) forms of RhoA, Rac1, and Cdc42 as tools to evaluate the precise contribution of each GTPase to epithelial structure and barrier function. All mutant GTPases induced time-dependent disruptions in epithelial gate function and distinct morphological alterations in apical and basal F-actin pools. TJ proteins occludin, ZO-1, claudin-1, claudin-2, and junctional adhesion molecule (JAM)-1 were dramatically redistributed in the presence of CA RhoA or CA Cdc42, whereas only claudins-1 and -2 were redistributed in response to CA Rac1. DN Rac1 expression also induced selective redistribution of claudins-1 and -2 in addition to JAM-1, whereas DN Cdc42 influenced only claudin-2 and DN RhoA had no effect. AJ protein localization was unaffected by any mutant GTPase, but DN Rac1 induced a reduction in E-cadherin detergent solubility. All CA GTPases increased the detergent solubility of claudins-1 and -2, but CA RhoA alone reduced claudin-2 and ZO-1 partitioning to detergent-insoluble membrane rafts. We conclude that Rho family GTPases regulate epithelial intercellular junctions via distinct morphological and biochemical mechanisms and that perturbations in barrier function reflect any imbalance in active/resting GTPase levels rather than simply loss or gain of GTPase activity. epithelium; tight junctions; paracellular permeability; Madin-Darby canine kidney cells     

Oncogenic Raf-1 disrupts epithelial tight junctions via downregulation of occludin

Occludin is an integral membrane protein of the epithelial cell tight junction (TJ). Its potential role in coordinating structural and functional events of TJ formation has been suggested recently. Using a rat salivary gland epithelial cell line (Pa-4) as a model system, we have demonstrated that occludin not only is a critical component of functional TJs but also controls the phenotypic changes associated with epithelium oncogenesis. Transfection of an oncogenic Raf-1 into Pa-4 cells resulted in a complete loss of TJ function and the acquisition of a stratified phenotype that lacked cell-cell contact growth control. The expression of occludin and claudin-1 was downregulated, and the distribution patterns of ZO-1 and E-cadherin were altered. Introduction of the human occludin gene into Raf-1-activated Pa-4 cells resulted in reacquisition of a monolayer phenotype and the formation of functionally intact TJs. In addition, the presence of exogenous occludin protein led to a recovery in claudin-1 protein level, relocation of the zonula occludens 1 protein (ZO-1) to the TJ, and redistribution of E-cadherin to the lateral membrane. Furthermore, the expression of occludin inhibited anchorage-independent growth of Raf-1-activated Pa-4 cells in soft agarose. Thus, occludin may act as a pivotal signaling molecule in oncogenic Raf- 1-induced disruption of TJs, and regulates phenotypic changes associated with epithelial cell transformation.     

Salmonella type III effector AvrA stabilizes cell tight junctions to inhibit inflammation in intestinal epithelial cells

Salmonella Typhimurium is a major cause of human gastroenteritis. The Salmonella type III secretory system secretes virulence proteins, called effectors. Effectors are responsible for the alteration of tight junction (TJ) structure and function in intestinal epithelial cells. AvrA is a newly described bacterial effector found in Salmonella. We report here that AvrA expression stabilizes cell permeability and tight junctions in intestinal epithelial cells. Cells colonized with an AvrA-deficient bacterial strain (AvrA-) displayed decreased cell permeability, disruption of TJs, and an increased inflammatory response. Western blot data showed that TJ proteins, such as ZO-1, claudin-1, decreased after AvrA- colonization for only 1 hour. In contrast, cells colonized with AvrA-sufficient bacteria maintained cell permeability with stabilized TJ structure. This difference was confirmed in vivo. Fluorescent tracer studies showed increased fluorescence in the blood of mice infected with AvrA- compared to those infected with the AvrA-sufficient strains. AvrA- disrupted TJ structure and function and increased inflammation in vivo, compared to the AvrA- sufficient strain. Additionally, AvrA overexpression increased TJ protein expression when transfected into colonic epithelial cells. An intriguing aspect of this study is that AvrA stabilized TJs, even though the other TTSS proteins, SopB, SopE, and SopE2, are known to disrupt TJs. AvrA may play a role in stabilizing TJs and balancing the opposing action of other bacterial effectors. Our findings indicate an important role for the bacterial effector AvrA in regulation of intestinal epithelial cell TJs during inflammation. The role of AvrA represents a highly refined bacterial strategy that helps the bacteria survive in the host and dampen the inflammatory response.     

Claudin-based tight junctions are crucial for the mammalian epidermal barrier: a lesson from claudin-1-deficient mice

The tight junction (TJ) and its adhesion molecules, claudins, are responsible for the barrier function of simple epithelia, but TJs have not been thought to play an important role in the barrier function of mammalian stratified epithelia, including the epidermis. Here we generated claudin-1-deficient mice and found that the animals died within 1 d of birth with wrinkled skin. Dehydration assay and transepidermal water loss measurements revealed that in these mice the epidermal barrier was severely affected, although the layered organization of keratinocytes appeared to be normal. These unexpected findings prompted us to reexamine TJs in the epidermis of wild-type mice. Close inspection by immunofluorescence microscopy with an antioccludin monoclonal antibody, a TJ-specific marker, identified continuous TJs in the stratum granulosum, where claudin-1 and -4 were concentrated. The occurrence of TJs was also confirmed by ultrathin section EM. In claudin-1-deficient mice, claudin-1 appeared to have simply been removed from these TJs, leaving occludin-positive (and also claudin-4-positive) TJs. Interestingly, in the wild-type epidermis these occludin-positive TJs efficiently prevented the diffusion of subcutaneously injected tracer (approximately 600 D) toward the skin surface, whereas in the claudin-1-deficient epidermis the tracer appeared to pass through these TJs. These findings provide the first evidence that continuous claudin-based TJs occur in the epidermis and that these TJs are crucial for the barrier function of the mammalian skin.