The claudin-like megatrachea is essential in septate junctions for the epithelial barrier function in Drosophila

Vertebrate claudin proteins are integral components of tight junctions, which function as paracellular diffusion barriers in epithelia. We identified Megatrachea (Mega), a Drosophila transmembrane protein homologous to claudins, and show that it acts in septate junctions, the corresponding structure of invertebrates. Our analysis revealed that Mega has transepithelial barrier function similar to the claudins. Also, Mega is necessary for normal tracheal cell morphogenesis but not for apicobasal polarity or epithelial integrity. In addition, we present evidence that Mega is essential for localization of the septate junction protein complex Coracle/Neurexin. The results indicate that claudin-like proteins are functionally conserved between vertebrates and Drosophila.     

Regulation of tight junctions and loss of barrier function in pathophysiology

The mechanism by which epithelial and endothelial cells interact to form polarized tissue is of fundamental importance to multicellular organisms. Dysregulation of these barriers occurs in a variety of diseases, destroying the normal cellular environments and leading to organ failure. Increased levels of growth factors are a common characteristic of diseases exhibiting tissue permeability, suggesting that growth factors play a direct role in elevating permeability. Of particular concern for this laboratory, increased expression of vascular endothelial growth factor may enhance vascular permeability in diabetic retinopathy, leading to vision impairment and blindness. However, the mechanism by which growth factors increase permeability is unclear. Polarized cells form strong barriers through the development of tight junctions, which are specialized regions of the junctional complex. Tight junctions are composed of three types of transmembrane proteins, a number of peripheral membrane structural proteins, and are associated with a variety of regulatory proteins. Recent data suggest that growth factor-stimulated alterations in tight junctions contribute to permeability in a variety of disease states. The goal of this review was to elucidate potential mechanisms by which elevated growth factors elicit deregulated paracellular permeability via altered regulation of tight junctions, with particular emphasis on the tight junction proteins occludin and ZO-1, protein kinase C signaling, and endocytosis of junctional proteins. Understanding the molecular mechanisms underlying growth factor-mediated regulation of tight junctions will facilitate the development of novel treatments for diseases such as brain tumors, diabetic retinopathy and other diseases with compromised tight junction barriers.     

Formation and barrier function of tight junctions in human ovarian surface epithelium

The normal ovarian surface epithelium (OSE) is a primitive epithelium made up by a single layer of mesothelial-type epithelial cells. When these cells get trapped in the ovarian stroma, expression of epithelial specific markers, such as E-cadherin, are induced. Most epithelial cells are also characterized by the ability to form tight junctions (TJ). Incomplete TJ have earlier been demonstrated in the OSE by electron microscopy studies. We have investigated expression and localization of the TJ proteins ZO-1, occludin, and claudin-1 in tissue biopsies from normal human ovaries and OSE in culture. The dynamics of TJ formation were studied in human OSE cultured on porous filters in culture inserts by measuring trans epithelial resistance (TER) including Ca(2+) switch experiments. Confluent OSE cells were also analyzed by electron microscopy. The results show that normal human OSE has expression of all three TJ proteins investigated. These proteins, ZO-1, occludin, and claudin-1, were localized to OSE cell borders both in ovarian biopsies and in cultured OSE. There was no difference in this regard between fertile and postmenopausal women. Cells in culture were polarized and presented junctional complexes seen by electron microscopy. In the Ca(2+) switch experiments, removing free Ca(2+) transiently, TER decreased significantly (P < 0.05) in the Ca(2+)-free group compared with nontreated OSE. TER was fully restored after 24 h. N-cadherin but not E-cadherin was expressed in the OSE and localized to the cell borders. We conclude that normal human OSE express and form functional TJ both in vivo and vitro. This report also describes a method to study the influence of ovarian-derived mediators on TJ in cultured OSE.     

Maintenance of the epithelial barrier in a bronchial epithelial cell line is dependent on functional E-cadherin local to the tight junctions

Tight junctions (TJ) are essential components of polarized epithelia, and E-cadherin is important for their formation and maintenance. The bronchial epithelial cell line, 16HBE14o- expresses E- and P-cadherin, but not N-cadherin. E- and P-cadherin levels changed during culture, the former increasing after confluence, and the latter were markedly reduced. All detectable E-cadherin was bound to β- and γ-catenins. We investigated involvement of E-cadherin with epithelial integrity using an E-cadherin specific, function-blocking antibody, SHE78-7. Surprisingly, apical SHE78-7 exposure caused a prompt fall in transepithelial resistance (TER), while TER remained unchanged for 8 hrs after basal exposure then dropped. SHE78-7 exposure increased epithelial permeability to mannitol, inulin, and 9.5 kDa and 77 kDa dextrans and caused fragmentation and loss of the tight junction protein, ZO-1, from the cell borders in some areas. Ultrastructural studies showed that all junctional intercellular contact was lost in the center of SHE78-7 induced lesions. Near the lesion periphery, epithelial structure was maintained, but TJs were dysfunctional as shown by ruthenium red penetration. Analysis of epithelial penetration by SHE78-7 revealed discrete, local defects in the apical barrier at the top of some cell hills that permitted rapid access of the antibody to E-cadherin near the apical surface. In contrast, after basal exposure, antibody initially engaged with E-cadherin nearer the basal surface and only accessed apical E-cadherin later. Taken together with the TER measurements, these data suggest compartmentalization of E-cadherin function within 16HBE14o- cells, with only the apical E-cadherin adjacent to the tight junctions contributing to the function of the latter.     

TNFAIP3 maintains intestinal barrier function and supports epithelial cell tight junctions

Tight junctions between intestinal epithelial cells mediate the permeability of the intestinal barrier, and loss of intestinal barrier function mediated by TNF signaling is associated with the inflammatory pathophysiology observed in Crohn's disease and celiac disease. Thus, factors that modulate intestinal epithelial cell response to TNF may be critical for the maintenance of barrier function. TNF alpha-induced protein 3 (TNFAIP3) is a cytosolic protein that acts in a negative feedback loop to regulate cell signaling induced by Toll-like receptor ligands and TNF, suggesting that TNFAIP3 may play a role in regulating the intestinal barrier. To investigate the specific role of TNFAIP3 in intestinal barrier function we assessed barrier permeability in TNFAIP3(-/-) mice and LPS-treated villin-TNFAIP3 transgenic mice. TNFAIP3(-/-) mice had greater intestinal permeability compared to wild-type littermates, while villin-TNFAIP3 transgenic mice were protected from increases in permeability seen within LPS-treated wild-type littermates, indicating that barrier permeability is controlled by TNFAIP3. In cultured human intestinal epithelial cell lines, TNFAIP3 expression regulated both TNF-induced and myosin light chain kinase-regulated tight junction dynamics but did not affect myosin light chain kinase activity. Immunohistochemistry of mouse intestine revealed that TNFAIP3 expression inhibits LPS-induced loss of the tight junction protein occludin from the apical border of the intestinal epithelium. We also found that TNFAIP3 deubiquitinates polyubiquitinated occludin. These in vivo and in vitro studies support the role of TNFAIP3 in promoting intestinal epithelial barrier integrity and demonstrate its novel ability to maintain intestinal homeostasis through tight junction protein regulation.     

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.     

Possible involvement of gap junctions in the barrier function of tight junctions of brain and lung endothelial cells

Gap-junction plaques are often observed with tight-junction strands of vascular endothelial cells but the molecular interaction and functional relationships between these two junctions remain obscure. We herein show that gap-junction proteins connexin40 (Cx40) and Cx43 are colocalized and coprecipitated with tight-junction molecules occludin, claudin-5, and ZO-1 in porcine blood-brain barrier (BBB) endothelial cells. Gap junction blockers 18beta-glycyrrhetinic acid (18beta-GA) and oleamide (OA) did not influence expression of Cx40, Cx43, occludin, claudin-5, junctional adhesion molecule (JAM)-A, JAM-B, JAM-C, or ZO-1, or their subcellular localization in the porcine BBB endothelial cells. In contrast, these gap-junction blocking agents inhibited the barrier function of tight junctions in cells, determined by measurement of transendothelial electrical resistance and paracellular flux of mannitol and inulin. 18beta-GA also significantly reduced the barrier property in rat lung endothelial (RLE) cells expressing doxycycline-induced claudin-1, but did not change the interaction between Cx43 and either claudin-1 or ZO-1, nor their expression levels or subcellular distribution. These findings suggest that Cx40- and/or Cx43-based gap junctions might be required to maintain the endothelial barrier function without altering the expression and localization of the tight-junction components analyzed.     

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.     

Occludin oligomeric assembly at tight junctions of the blood-brain barrier is disrupted by peripheral inflammatory hyperalgesia

Tight junctions (TJs) at the blood-brain barrier (BBB) dynamically alter paracellular diffusion of blood-borne substances from the peripheral circulation to the CNS in response to external stressors, such as pain, inflammation, and hypoxia. In this study, we investigated the effect of lambda-carrageenan-induced peripheral inflammatory pain (i.e., hyperalgesia) on the oligomeric assembly of the key TJ transmembrane protein, occludin. Oligomerization of integral membrane proteins is a critical step in TJ complex assembly that enables the generation of tightly packed, large multiprotein complexes capable of physically obliterating the interendothelial space to inhibit paracellular diffusion. Intact microvessels isolated from rat brains were fractionated by detergent-free density gradient centrifugation, and gradient fractions were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis/ Western blot. Injection of lambda-carrageenan into the rat hind paw produced after 3 h a marked change in the relative amounts of oligomeric, dimeric, and monomeric occludin isoforms associated with different plasma membrane lipid raft domains and intracellular compartments in endothelial cells at the BBB. Our findings suggest that increased BBB permeability (i.e., leak) associated with lambda-carrageenan-induced peripheral inflammatory pain is promoted by the disruption of disulfide-bonded occludin oligomeric assemblies, which renders them incapable of forming an impermeant physical barrier to paracellular transport.     

Kiwifruit cysteine protease actinidin compromises the intestinal barrier by disrupting tight junctions

BackgroundThe intestinal epithelium forms a barrier that food allergens must cross in order to induce sensitization. The aim of this study was to evaluate the impact of the plant-derived food cysteine protease — actinidin (Act d1) on the integrity of intestinal epithelium tight junctions (TJs).MethodsEffects of Act d1 on the intestinal epithelium were evaluated in Caco-2 monolayers and in a mouse model by measuring transepithelial resistance and in vivo permeability. Integrity of the tight junctions was analyzed by confocal microscopy. Proteolysis of TJ protein occludin was evaluated by mass spectrometry.ResultsActinidin (1 mg/mL) reduced the transepithelial resistance of the cell monolayer by 18.1% (after 1 h) and 25.6% (after 4 h). This loss of barrier function was associated with Act d 1 disruption of the occludin and zonula occludens (ZO)-1 network. The effect on intestinal permeability in vivo was demonstrated by the significantly higher concentration of 40 kDa FITC-dextran (2.33 μg/mL) that passed from the intestine into the serum of Act d1 treated mice in comparison to the control group (0.5 μg/mL). Human occludin was fragmented, and putative Act d1 cleavage sites were identified in extracellular loops of human occludin.ConclusionAct d1 caused protease-dependent disruption of tight junctions in confluent Caco-2 cells and increased intestinal permeability in mice.General significanceIn line with the observed effects of food cysteine proteases in occupational allergy, these results suggest that disruption of tight junctions by food cysteine proteases may contribute to the process of sensitization in food allergy.     

Ena/VASP is required for endothelial barrier function in vivo

Enabled/vasodilator-stimulated phosphoprotein (Ena/VASP) proteins are key actin regulators that localize at regions of dynamic actin remodeling, including cellular protrusions and cell–cell and cell–matrix junctions. Several studies have suggested that Ena/VASP proteins are involved in the formation and function of cellular junctions. Here, we establish the importance of Ena/VASP in endothelial junctions in vivo by analysis of Ena/VASP-deficient animals. In the absence of Ena/VASP, the vasculature exhibits patterning defects and lacks structural integrity, leading to edema, hemorrhaging, and late stage embryonic lethality. In endothelial cells, we find that Ena/VASP activity is required for normal F-actin content, actomyosin contractility, and proper response to shear stress. These findings demonstrate that Ena/VASP is critical for actin cytoskeleton remodeling events involved in the maintenance of functional endothelia.     

The acyl-CoA binding protein is required for normal epidermal barrier function in mice

The acyl-CoA binding protein (ACBP) is a 10 kDa intracellular protein expressed in all eukaryotic species. Mice with targeted disruption of Acbp (ACBP(-/-) mice) are viable and fertile but present a visible skin and fur phenotype characterized by greasy fur and development of alopecia and scaling with age. Morphology and development of skin and appendages are normal in ACBP(-/-) mice; however, the stratum corneum display altered biophysical properties with reduced proton activity and decreased water content. Mass spectrometry analyses of lipids from epidermis and stratum corneum of ACBP(+/+) and ACBP(-/-) mice showed very similar composition, except for a significant and specific decrease in the very long chain free fatty acids (VLC-FFA) in stratum corneum of ACBP(-/-) mice. This finding indicates that ACBP is critically involved in the processes that lead to production of stratum corneum VLC-FFAs via complex phospholipids in the lamellar bodies. Importantly, we show that ACBP(-/-) mice display a ～50% increased transepidermal water loss compared with ACBP(+/+) mice. Furthermore, skin and fur sebum monoalkyl diacylglycerol (MADAG) levels are significantly increased, suggesting that ACBP limits MADAG synthesis in sebaceous glands. In summary, our study shows that ACBP is required for production of VLC-FFA for stratum corneum and for maintaining normal epidermal barrier function.     

Thr203 of claudin-1, a putative phosphorylation site for MAP kinase, is required to promote the barrier function of tight junctions

Mitogen-activated protein kinase (MAPK) modulates the barrier function of tight junctions. We identified a putative phosphorylation site for MAPK at around Thr203 (PKPTP) in claudin-1, and determined the biological significance of this site. To this end, using the rat lung endothelial cell line RLE, we generated cells expressing doxycycline (Dox)-inducible wild-type claudin-1 and its mutant with substitution of Thr203 to Ala, and named them RLE:rtTA:CL1 and RLE:rtTA:CL1T203A, respectively. We herein show, by measurement of transendothelial electrical resistance and paracellular flux of mannitol and inulin, that functional tight junctions were reconstituted in both cells by Dox-induced expression of claudin-1. Interestingly, the barrier functions of tight junctions were less developed in RLE:rtTA:CL1T203A cells compared with RLE:rtTA:CL1 cells. Consistently, levels of both detergent-insoluble claudin-1 protein and its threonine-phosphorylation after Dox treatment were low in RLE:rtTA:CL1T203A cells compared to RLE:rtTA:CL1 cells. Furthermore, pretreatment with the MAPK inhibitor PD98059 markedly suppressed the barrier function and amount of detergent-insoluble claudin-1 in Dox-exposed RLE:rtTA:CL1 cells, whereas it marginally influenced those in RLE:rtTA:CL1T203A cells. These findings indicate that Thr203 of claudin-1 is required to enhance the barrier function of claudin-1-based tight junctions, probably via its phosphorylation and subsequent integration into tight junctions.     

Tjp3/zo-3 is critical for epidermal barrier function in zebrafish embryos

TJP3/ZO-3 is a scaffolding protein that tethers tight junction integral membrane proteins to the actin cytoskeleton and links the conserved Crumbs polarity complex to tight junctions. The physiological function of TJP3/ZO-3 is not known and mice lacking TJP3/ZO-3 show no apparent phenotype. Here we show that Tjp3/Zo-3 is a component of tight junctions present in the enveloping cell layer of zebrafish embryos. Silencing tjp3/zo-3 using morpholinos leads to edema, loss of blood circulation and tail fin malformations in the embryos. The ultrastructure of tight junctions of the enveloping cell layer is disrupted, without affecting the asymmetric distribution of plasma membrane proteins. Morphants show a loss of the epidermal barrier, as assessed by an increased permeability of the enveloping cell layer to low molecular weight tracers and a higher sensitivity of the embryos to osmotic stress. Subjecting wild-type embryos to osmotic stress mimicks the morphant phenotype, consistent with the phenotype being a direct consequence of failed osmoregulation. Thus, Tjp3/Zo-3 is critical for barrier function of the enveloping cell layer and osmoregulation in early stages of zebrafish development.     

In vivo assembly of tight junctions in fetal rat liver

Examination of glutaraldehyde-fixed, freeze-fractured livers from 14-15-day rat fetuses provided the basis for the following observations. Membrane particles align in otherwise poorly particulated areas of the presumptive pericanalicular plasma membrane (A face), frequently forming a discontinuous "honey-comb" network joining small particle islands. Even at this early stage, contiguous B-fracture faces contain furrows, rather than rows of pits, distinguishing the linear particle aggregates on the A face as developing tight junctions rather than gap junctions. Short segments of these linear arrays merge with smooth ridges clearly identifiable as segments of discontinuous tight junctions. With the continuing confluence of particulate and smooth ridge segments, mature tight junctions become fully appreciable. We conclude that tight junctions form de novo by the alignment and fusion of separate particles into beaded ridges which, in turn, become confluent and are transformed into continuous smooth ones. At 21 days of fetal life, most of the images of assembly have disappeared, and the liver reveals well-formed bile canaliculi sealed by mature tight junctions.     

The coxsackie- and adenovirus receptor (CAR) is an in vivo marker for epithelial tight junctions, with a potential role in regulating permeability and tissue homeostasis

The coxsackie- and adenovirus receptor (CAR) is a transmembrane protein belonging to the immunoglobulin superfamily. The function of CAR as a virus receptor has been extensively analyzed, while its physiological role and expression pattern in adult tissues have remained less clear. CAR associates with epithelial tight junctions in vitro and mediates cell-cell adhesion. Using a set of affinity-purified antibodies, we show that CAR is predominantly expressed in epithelial cells lining the body cavities in adult mice, where it specifically co-localizes with the tight junction components ZO-1 and occludin. Notably, CAR could not be detected in endothelial cells of the vasculature, including brain capillaries. CAR expression correlated positively with the maturity of tight junctions and inversely with permeability. With a few exceptions, the two known CAR isoforms were co-expressed in most epithelial cells analyzed. A CAR mutant lacking the intracellular tail over-expressed in transgenic mice was diffusely localized over the plasma membrane, showing the importance of this domain for correct subcellular localization in vivo. We conclude that CAR is localized to epithelial tight junctions in vivo where it may play a role in the regulation of epithelial permeability and tissue homeostasis.