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.     

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.     

Direct association of occludin with ZO-1 and its possible involvement in the localization of occludin at tight junctions

Occludin is an integral membrane protein localizing at tight junctions (TJ) with four transmembrane domains and a long COOH-terminal cytoplasmic domain (domain E) consisting of 255 amino acids. Immunofluorescence and laser scan microscopy revealed that chick full- length occludin introduced into human and bovine epithelial cells was correctly delivered to and incorporated into preexisting TJ. Further transfection studies with various deletion mutants showed that the domain E, especially its COOH-terminal approximately 150 amino acids (domain E358/504), was necessary for the localization of occludin at TJ. Secondly, domain E was expressed in Escherichia coli as a fusion protein with glutathione-S-transferase, and this fusion protein was shown to be specifically bound to a complex of ZO-1 (220 kD) and ZO-2 (160 kD) among various membrane peripheral proteins. In vitro binding analyses using glutathione-S-transferase fusion proteins of various deletion mutants of domain E narrowed down the sequence necessary for the ZO-1/ZO-2 association into the domain E358/504. Furthermore, this region directly associated with the recombinant ZO-1 produced in E. coli. We concluded that occludin itself can localize at TJ and directly associate with ZO-1. The coincidence of the sequence necessary for the ZO-1 association with that for the TJ localization suggests that the association with underlying cytoskeletons through ZO-1 is required for occludin to be localized at TJ.     

Contribution of claudin-5 to barrier properties in tight junctions of epithelial cells

Claudin-5 is a transmembrane protein reported to be primarily present in tight junctions of endothelia. Unexpectedly, we found expression of claudin-5 in HT-29/B6 cells, an epithelial cell line derived from human colon. Confocal microscopy showed colocalization of claudin-5 with occludin, indicating its presence in the tight junctions. By contrast, claudin-5 was absent in the human colonic cell line Caco-2 and in Madin-Darby canine kidney cells (MDCK sub-clones C7 and C11), an epithelial cell line derived from the collecting duct. To determine the contribution of claudin-5 to tight junctional permeability in cells of human origin, stable transfection of Caco-2 with FLAG-claudin-5 cDNA was performed. In addition, clone MDCK-C7 was transfected. Synthesis of the exogenous FLAG-claudin-5 was verified by Western blot analysis and confocal fluorescent imaging by employing FLAG-specific antibody. FLAG-claudin-5 was detected in transfected cells in colocalization with occludin, whereas cells transfected with the vector alone did not exhibit specific signals. Resistance measurements and mannitol fluxes after stable transfection with claudin-5 cDNA revealed a marked increase of barrier function in cells of low genuine transepithelial resistance (Caco-2). By contrast, no changes of barrier properties were detected in cells with a high transepithelial resistance (MDCK-C7) after stable transfection with claudin-5 cDNA. We conclude that claudin-5 is present in epithelial cells of colonic origin and that it contributes to some extent to the paracellular seal. Claudin-5 may thus be classified as a tight-junctional protein capable of contributing to the "sealing" of the tight junction.     

Enteropathogenic Escherichia coli dephosphorylates and dissociates occludin from intestinal epithelial tight junctions

Enteropathogenic Escherichia coli (EPEC) increases tight junction permeability in part by phosphorylating the 20 kDa myosin light chain (MLC₂₀) that induces cytoskeletal contraction. The impact of this enteric pathogen on specific tight junction (TJ) proteins has not been investigated. We examined the effect of EPEC infection on occludin localization and phosphorylation in intestinal epithelial cells. After infection by EPEC, a progressive shift of occludin from a primarily TJ-associated domain to an intracellular compartment occurred, as demonstrated by immunofluorescent staining. A reverse in the ratio of phosphorylated to dephosphorylated occludin accompanied this morphological change. Eradication of EPEC with gentamicin resulted in the normalization of occludin localization and phosphorylation. The serine/threonine phosphatase inhibitor, calyculin A, prevented these events. The EPEC-associated decrease in transepithelial electrical resistance, a measure of TJ barrier function, returned to baseline after gentamicin treatment. Non-pathogenic E. coli , K-12, did not induce these changes. Transformation of K-12 with the pathogenicity island of EPEC, however, conferred the phenotype of wild-type EPEC. Deletion of specific EPEC genes encoding proteins involved in EPEC type III secretion markedly attenuated these effects. These findings suggest that EPEC-induced alterations in occludin contribute to the pathophysiology associated with this infection.     

Decreased interaction between ZO-1 and occludin is involved in alteration of tight junctions in transplanted epiphora submandibular glands

Tight junctions (TJs) in salivary epithelium play an important role in regulating saliva secretion. Autologous transplantation of submandibular glands (SMGs) is an effective method to treat severe dry eye syndrome. However, epiphora occurs in some patients 6 months after transplantation. We previously found that the acinar TJs are enlarged in rabbit SMGs after long-term transplantation, but the exact TJ components involved in the epiphora are still unknown. Here, we found that the mRNA and protein expression of ZO-1 and occludin were increased in the transplanted SMGs obtained from epiphora patients, while other TJs were unchanged. The intensity of ZO-1 and occludin at the apicolateral membranes as well as occludin in the cytoplasm were increased in epiphora SMGs, but the interaction between ZO-1 and occludin was decreased as evidenced by both co-immunoprecipitation assay and co-immunofluorescence staining. Mechanically, the expression of casein kinase 2α (CK2α) and CK2β, which was reported to affect occludin modification and the interaction of occludin with ZO-1 in previous literatures, were increased in epiphora glands. Moreover, activation of muscarinic acetylcholine receptor (mAChR) by carbachol directly decreased the interaction between ZO-1 and occludin and increased the acinar TJ width in the freshly isolated human SMGs, whereas these effects were abolished by pretreatment with CK2 inhibitor. Taken together, our findings suggest that decreased interaction between ZO-1 and occludin might contribute to the epiphora occurred in the transplanted SMGs, and mAChR together with the intracellular molecule CK2 might be responsible for the alteration of TJs in epiphora glands.     

Aldosterone and tight junctions: modulation of claudin-4 phosphorylation in renal collecting duct cells

Aldosterone classically modulates Na transport in tight epithelia such as the renal collecting duct (CD) through the transcellular route, but it is not known whether the hormone could also affect paracellular permeability. Such permeability is controlled by tight junctions (TJ) that form a size- and charge-selective barrier. Among TJ proteins, claudin-4 has been highlighted as a key element to control paracellular charge selectivity. In RCCD2 CD cells grown on filters, we have identified novel early aldosterone effects on TJ. Endogenous claudin-4 abundance and cellular localization were unaltered by aldosterone. However, the hormone promoted rapid (within 15-20 min) and transient phosphorylation of endogenous claudin-4 on threonine residues, without affecting tyrosine or serine; this event was fully developed at 10 nM aldosterone and appeared specific for aldosterone (because it is not observed after dexamethasone treatment and it depends on mineralocorticoid receptor occupancy). Within the same delay, aldosterone also promoted an increased apical-to-basal passage of 125I (a substitute for 36Cl), whereas 22Na passage was unaffected; paracellular permeability to [3H]mannitol was also reduced. Later on (45 min), a fall in transepithelial resistance was observed. These data indicate that aldosterone modulates TJ properties in renal epithelial cells.     

Immunolocalization of tight junction proteins in blood vessels in human germinal matrix and cortex

Brain development occurs in a specialized environment maintained by a blood–brain barrier (BBB). An important structural element of the BBB is the endothelial tight junction (TJ). TJs are present during the embryonic period, but BBB impermeability accrues over an extended gestational interval. In studies of human premature infants, we used immunomicroscopy to determine if amounts of the TJ proteins ZO-1, claudin and occludin increase with gestational age in vessels of germinal matrix (GM) and cortex. By 24 weeks postconception (PC), TJ proteins were present in both GM and cortical vessels, but immunoreactivity in the GM of the youngest subjects was less than in older subjects. At 24 weeks PC, TJ protein immunoreactivity in GM vessels was less than in cortical vessels suggesting that TJ maturation progresses along a superficial to deep brain axis. This concept correlates with conclusions from previous analyses of the expression of brain endothelial cell alkaline phosphatase (AP) activity. AP appears in cortical vessels before appearing in deep white matter and GM vessels. Together, these data indicate that differentiation of some functional specializations is still in progress in GM vessels during the third trimester. This maturation could relate to the pathogenesis of germinal matrix hemorrhage–intraventricular hemorrhage.     

Activin A and follicle-stimulating hormone control tight junctions in avian granulosa cells by regulating occludin expression

Within the avian ovarian follicle, the oocyte is surrounded by a monolayer of granulosa cells, which exhibit pronounced epithelial properties. Here we demonstrate the presence of the major tight junction protein occludin in granulosa cells. As shown by immunohistochemistry, occludin localizes to the oocyte-facing granulosa cell surface. Occludin and thus tight junctions are dynamically regulated in a developmental stage-specific manner. Small white follicles, which have not yet started yellow yolk incorporation, show pronounced occludin expression in vitro and in vivo. By contrast, yellow yolk-incorporating small yellow follicles exhibit much lower levels of occludin, and hierarchical, preovulatory follicles are virtually devoid of this essential tight junction component. Using a primary granulosa cell culture system, we demonstrate that concerted action of two well-established ovarian growth regulators, follicle-stimulating hormone and activin A, leads to strong induction of occludin expression in vitro. We suggest that the stage-dependent decrease in the granulosa cell growth factor responsiveness triggers the disruption of tight junctions, enabling rapid and high capacity transport of macromolecules into the oocyte through a paracellular pathway. Such a high-capacity transport for yolk components may represent a crucial prerequisite for rapid oocyte growth once follicles have entered the follicular hierarchy.     

Enhancement of barrier function by overexpression of claudin-4 in tight junctions of submandibular gland cells

In salivary glands, primary saliva is produced by acini and is modified by the reabsorption and secretion of ions in the ducts. Thus, the permeability of intercellular junctions in the ducts is considered to be lower than in the acini. We have examined the relationship between the expressed claudin isotypes and the barrier functions of tight junctions in a submandibular gland epithelial cell line, SMIE. SMIE cells were originally derived from rat submandibular duct cells, but their barrier functions are not as efficient as those of Madin-Darby canine kidney cells. Large molecules, such as 70-kDa dextran, diffuse across the monolayers, although E-cadherin and occludin, adherens junction and tight junction proteins, respectively, are expressed in SMIE cells. Claudin-3 protein has also been detected, but the expression level of claudin-3 mRNA is much lower than in the original submandibular glands. Other claudins including claudin-4 (originally expressed in the duct cells) have not been detected. Because of the limited expression of claudins, SMIE cells are suitable for studying the role(s) of claudins. To examine the function of claudin-4 in submandibular glands, we have overexpressed green fluorescence protein (GFP)-fused claudin-4 in SMIE cells. Cells that express GFP-fused claudin-4 have a higher transepithelial electrical resistance and a lower permeability of 70-kDa dextran, although the expression levels of occludin and claudin-3 are hardly affected. Therefore, claudin-4 plays a role in the regulation of the barrier function of tight junctions in submandibular glands. This work was supported by Grants-in-Aid for scientific research from the Ministry of Education, Science, Culture, Sports, and Technology of Japan (16591868), by a Nihon University Multidisciplinary Research Grant for 2006 and 2007, and by a Grant-in-Aid for a 2003 Multidisciplinary Research Project from MEXT.     

Mutated C-terminal fragments of Clostridium perfringens enterotoxin have increased affinity to claudin-4 and reversibly modulate tight junctions in vitro

Passage across epithelial cell sheets is the first step in drug absorption. Tight junctions (TJs) are located between adjacent epithelial cells and seal the intercellular space preventing leakage of solutes. Claudin, a tetra-transmembrane protein family, is a pivotal functional and structural component of the TJ barrier. Modulation of the claudin-based TJ seal is a strategy for mucosal drug absorption. We previously found that a claudin-4 binder, a C-terminal fragment of Clostridium perfringens enterotoxin (C-CPE194), was a modulator of the TJ seal and a potent mucosal absorption enhancer. In the present study, we attempted to improve claudin-4 binders by modification of C-CPE194. Substitution of Asn at position 309 and Ser at position 313 with Ala increased the affinity to claudin-4 by 9.9-fold as compared to C-CPE194. Deletion of 10 amino acids in the N-terminal domain of the double-alanine-substituted mutant increased affinity to claudin-4 by 23.9-fold as compared to C-CPE194. These C-CPE194 mutants reversibly modulated the TJ seal in human intestinal epithelial cell sheets. The N-terminal-truncated mutant was the most potent modulator of the TJ seal. These findings indicate that the C-CPE mutant may be a promising lead for the development of a clinical TJ modulator.     

CNS myelin and sertoli cell tight junction strands are absent in Osp/claudin-11 null mice

Oligodendrocyte-specific protein (OSP)/claudin-11 is a recently identified transmembrane protein found in CNS myelin and testis with unknown function. Herein we demonstrate that Osp null mice exhibit both neurological and reproductive deficits: CNS nerve conduction is slowed, hindlimb weakness is conspicuous, and males are sterile. Freeze fracture reveals that tight junction intramembranous strands are absent in CNS myelin and between Sertoli cells of mutant mice. Our results demonstrate that OSP is the mediator of parallel-array tight junction strands and distinguishes this protein from other intrinsic membrane proteins in tight junctions. These novel results provide direct evidence of the pivotal role of the claudin family in generating the paracellular physical barrier of tight junctions necessary for spermatogenesis and normal CNS function.     

Signalling to and from tight junctions

Tight junctions have long been regarded as simple barriers that separate compartments of different compositions, but recent research indicates that different types of signalling proteins and transduction pathways are associated with these junctions. They receive and convert signals from the cell interior to regulate junction assembly and function, and transmit signals to the cell interior to modulate gene expression and cell behaviour.     

Protein phosphatases 2A and 1 interact with occludin and negatively regulate the assembly of tight junctions in the CACO-2 cell monolayer

Occludin is hyperphosphorylated on Ser and Thr residues in intact epithelial tight junction (TJ); however, the role of this phosphorylation in the assembly of TJ is unclear. The influence of protein phosphatases PP2A and PP1 on the assembly of TJ and phosphorylation of occludin was evaluated in Caco-2 cells. Protein phosphatase inhibitors and reduced expression of PP2A-Calpha and PP1alpha accelerated the calcium-induced increase in transepithelial electrical resistance and barrier to inulin permeability and also enhanced the junctional organization of occludin and ZO-1 during TJ assembly. Phosphorylation of occludin on Thr residues, but not on Ser residues, was dramatically reduced during the disassembly of TJ and was gradually increased during the reassembly. PP2A and PP1 co-immunoprecipitate with occludin, and this association was reduced during the assembly of TJ. Glutathione S-transferase (GST) pull-down assay using recombinant GST-occludin demonstrated that cellular PP2A and PP1 bind to the C-terminal tail of occludin, and these interactions were also reduced during the assembly of TJ. A pairwise binding assay using GST-occludin and purified PP2A and PP1 demonstrates that PP2A and PP1 directly interacts with the C-terminal tail of occludin. In vitro incubation of phospho-occludin with PP2A or PP1 indicated that PP2A dephosphorylates occludin on phospho-Thr residues, whereas PP1 dephosphorylates it on phospho-Ser. This study shows that PP2A and PP1 directly interact with occludin and negatively regulate the assembly of TJ by modulating the phosphorylation status of occludin.     

Coxsackievirus entry across epithelial tight junctions requires occludin and the small GTPases Rab34 and Rab5

The major group B coxsackievirus (CVB) receptor is a component of the epithelial tight junction (TJ), a protein complex that regulates the selective passage of ions and molecules across the epithelium. CVB enters polarized epithelial cells from the TJ, causing a transient disruption of TJ integrity. Here we show that CVB does not induce major reorganization of the TJ, but stimulates the specific internalization of occludin-a TJ integral membrane component-within macropinosomes. Although occludin does not interact directly with virus, depletion of occludin prevents CVB entry into the cytoplasm and inhibits infection. Both occludin internalization and CVB entry require caveolin but not dynamin; both are blocked by inhibitors of macropinocytosis and require the activity of Rab34, Ras, and Rab5, GTPases known to regulate macropinocytosis. Thus, CVB entry depends on occludin and occurs by a process that combines aspects of caveolar endocytosis with features characteristic of macropinocytosis.     

1-Naphthylisothiocyanate-induced permeability of hepatic tight junctions to proteins.

We have studied the early action of 1-naphthylisothiocyanate (ANIT) in relation to its effect on the permeability barrier formed by hepatic tight junctions. Materials having different Mr values [inulin (5000), horseradish peroxidase (HRP) (40,000), ovalbumin (also 40,000) and pig gamma-globulin (IgG) (160,000)] were individually pulsed, within 1 min, into perfused rat livers operating under single-pass conditions. In untreated rats, a small peak of HRP and ovalbumin and a comparatively larger peak of inulin were observed in the bile at 7 min. In rats treated with ANIT, with increasing duration of ANIT treatment the inulin peak increased proportionally, whereas the HRP and ovalbumin peaks remained unchanged until after 10 h of ANIT exposure; gamma-globulin was not detected in the 7 min bile sample until after 14 h of ANIT treatment. Bile flow in all rats remained approximately the same until after 14 h of ANIT pretreatment, when substantial bile-flow reduction was observed. Phenobarbitone pretreatment increased the effect of ANIT and massively elevated the first HRP peak; it also shortened the time (to 4 h) at which the increase in permeability to this protein was observed. In contrast, the first HRP peak was virtually abolished in rats that had received the mixed-function-oxidase inhibitor SKF 525A. These experiments suggest that (i) ANIT progressively increased the permeability of the junctional barrier before the reduction in bile flow, (ii) the ANIT-increased permeability change seems to be inversely dependent upon the Mr of the infused proteins, and (iii) metabolites of ANIT were involved in the development of the junctional permeability change.     

Phosphorylation of occludin correlates with occludin localization and function at the tight junction

Multiple forms of occludin were found in Madin-Darby caninekidney (MDCK) cells. In the absence of cell-to-cell contacts, achievedby incubating cells in low-calcium growth medium, a cluster oflower-molecular-weight (LMW) occludin bands (~65,000-68,000) waspresent in both MDCK I and II cells. On formation of tight junctions,achieved by changing the low-calcium growth medium to normal-calciumgrowth medium, a cluster of higher-molecular-weight (HMW) bands(~72,000-75,000 for MDCK I cells and ~70,000-73,000 forMDCK II cells) was also expressed. The HMW occludin bands could beeliminated by phosphatase treatment. Therefore, the HMW forms ofoccludin appeared to be the hyperphosphorylated product of the LMWforms. These HMW forms were Triton X-100 insoluble, which correlatedwith their localization at the tight junctions. Furthermore, depletionof tight junction-localized occludin by an occludin extracellulardomian peptide (20) correlated with a decrease in the HMW forms ofoccludin. In conclusion, phosphorylation of occludin may be a mechanismby which occludin localization and function are regulated.

     

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.