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PubMed2000

Complex phenotype of mice lacking occludin, a component of tight junction strands.

Occludin is an integral membrane protein with four transmembrane domains that is exclusively localized at tight junction (TJ) strands. Here, we describe the generation and analysis of mice carrying a null mutation in the occludin gene. Occludin -/- mice were born with no gross phenotype in the expected Mendelian ratios, but they showed significant postnatal growth retardation. Occludin -/- males produced no litters with wild-type females, whereas occludin -/- females produced litters normally when mated with wild-type males but did not suckle them. In occludin -/- mice, TJs themselves did not appear to be affected morphologically, and the barrier function of intestinal epithelium was normal as far as examined electrophysiologically. However, histological abnormalities were found in several tissues, i.e., chronic inflammation and hyperplasia of the gastric epithelium, calcification in the brain, testicular atrophy, loss of cytoplasmic granules in striated duct cells of the salivary gland, and thinning of the compact bone. These phenotypes suggested that the functions of TJs as well as occludin are more complex than previously supposed.

Animals↗
PubMed2005

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.

Aldosterone↗
PubMed1998

Claudin-1 and -2: novel integral membrane proteins localizing at tight junctions with no sequence similarity to occludin.

Occludin is the only known integral membrane protein localizing at tight junctions (TJ), but recent targeted disruption analysis of the occludin gene indicated the existence of as yet unidentified integral membrane proteins in TJ. We therefore re-examined the isolated junction fraction from chicken liver, from which occludin was first identified. Among numerous components of this fraction, only a broad silver-stained band approximately 22 kD was detected with the occludin band through 4 M guanidine-HCl extraction as well as sonication followed by stepwise sucrose density gradient centrifugation. Two distinct peptide sequences were obtained from the lower and upper halves of the broad band, and similarity searches of databases allowed us to isolate two full-length cDNAs encoding related mouse 22-kD proteins consisting of 211 and 230 amino acids, respectively. Hydrophilicity analysis suggested that both bore four transmembrane domains, although they did not show any sequence similarity to occludin. Immunofluorescence and immunoelectron microscopy revealed that both proteins tagged with FLAG or GFP were targeted to and incorporated into the TJ strand itself. We designated them as "claudin-1" and "claudin-2", respectively. Although the precise structure/function relationship of the claudins to TJ still remains elusive, these findings indicated that multiple integral membrane proteins with four putative transmembrane domains, occludin and claudins, constitute TJ strands.

Amino Acid Sequence↗
PubMed2003

A dual-pathway ultrastructural model for the tight junction of rat proximal tubule epithelium.

A dual-pathway model is proposed for transport across the tight junction (TJ) in rat proximal tubule: large slit breaks formed by infrequent discontinuities in the TJ complex and numerous small circular pores, with spacing similar to that of claudin-2. This dual-pathway model is developed in the context of a proximal tubule model (Weinstein AM. Am J Physiol Renal Fluid Electrolyte Physiol 247: F848-F862, 1984) to provide an ultrastructural view of solute and water fluxes. Tubule model paramters (TJ reflection coefficient and water permeability), plus the measured epithelial NaCl and sucrose permeabilities, provide constraints for the dual-pathway model, which yields the small-pore radius and spacing and large slit height and area. For a small-pore spacing of 20.2 nm, comparable to the distance between adjacent particle pairs in apposing TJ strands, the small-pore radius is 0.668 nm and the large slit breaks have a height of 19.6 nm, occupying 0.04% of the total TJ length. This pore/slit geometry also satisfies the measured permeability for mannitol. The numerous small circular pores account for 91.25% of TJ NaCl permeability but only 5.0% of TJ water permeability. The infrequent large slit breaks in the TJ account for 95.0% of TJ water permeability but only 8.7% of TJ NaCl permeability. Sucrose and mannitol (4.6- and 3.6-A radius) can pass through both the large slit breaks and the small pores. For sucrose, 78.3% of the flux is via the slits and 21.7% via the pores; for mannitol, the flux is split nearly evenly between the two pathways, 50.8 and 49.2%. In this ultrastructural model, the TJ water permeability is 21.2% of the entire transepithelial water permeability and thus an order of magnitude greater than that predicted by the single-pore/slit theory (Preisig PA and Berry CA. Am J Physiol Renal Fluid Electrolyte Physiol 249: F124-F131, 1985).

Animals↗
PubMed2000

Corneal epithelial tight junctions and their response to lipopolysaccharide challenge.

PURPOSE: To investigate the expression and cellular distribution of putative tight junction (TJ) proteins occludin, ZO-1, ZO-2, and claudin-1 in rat corneal epithelium and alterations of TJs in cultured human corneal epithelial cells in response to lipopolysaccharide (LPS) challenge. METHODS: Immunohistochemistry was used to determine tissue distribution of occludin, ZO-1, ZO-2, and claudin-1 in the rat cornea. Reverse transcription-polymerase chain reaction was used to reveal the expression of mRNAs for claudins in simian virus (SV)40-immortalized human corneal epithelial (THCE) cells. To assess epithelial response to LPS challenge, THCE cells were cultured on the upper chamber of Transwell filters (Costar, Cambridge, MA), transepithelial electrical resistance (TER) was measured using a voltohmmeter. Immunocytochemistry and immunoblotting were used to assess alteration in the levels and localization of TJ-associated proteins occludin, ZO-1, and ZO-2 in LPS-treated THCE cells. RESULTS: Occludin, ZO-1, and ZO-2 were found at the cell borders of the superficial layer, whereas claudin-1 was localized mainly in the basal and wing cell layers of rat corneal epithelium. In addition to claudin-1, the transcripts for several other isotypes of claudins-2, -3, -7, -9, -14, and -15 were identified in THCE cells. Treatment of cultured THCE cells with LPS caused a dose- and time-dependent increase in monolayer permeability as assessed by TER measurements. The maximal decrease of TER was observed at approximately 6 to 9 hours after LPS challenge. The TER was then recovered gradually and returned to baseline after 24 hours. Examination of specific proteins associated with TJs by immunoblot analysis and immunomicroscopy revealed changes in the expression levels and localization of some of these proteins after their exposure to LPS. Specifically, LPS challenge resulted in a decrease in the levels of ZO-1 and ZO-2 compared with untreated cells. Reduction of the ZO-2 level was associated with the disappearance of ZO-2 staining from cell borders in 6-hour LPS-treated cells. CONCLUSIONS: Occludin, ZO-1, and ZO-2, but not claudin-1, are components of corneal epithelial TJs. LPS induces breakdown of the epithelial barrier through disruption of TJs, and ZO-1 and ZO-2 are targets for the induction.

Animals↗
PubMed2003

Astrocyte mediated modulation of blood-brain barrier permeability does not correlate with a loss of tight junction proteins from the cellular contacts.

In the central nervous system (CNS) complex endothelial tight junctions (TJs) form a restrictive paracellular diffusion barrier, the blood-brain barrier (BBB). Pathogenic changes within the CNS are frequently accompanied by the loss of BBB properties, resulting in brain edema. In order to investigate whether BBB leakiness can be monitored by a loss of TJ proteins from cellular borders, we used an in vitro BBB model where brain endothelial cells in co-culture with astrocytes form a tight permeability barrier for 3H-inulin and 14C-sucrose. Removal of astrocytes from the co-culture resulted in an increased permeability to small tracers across the brain endothelial cell monolayer and an opening of the TJs to horseradish peroxidase as detected by electron microscopy. Strikingly, opening of the endothelial TJs was not accompanied by any visible change in the molecular composition of endothelial TJs as junctional localization of the TJ-associated proteins claudin-3, claudin-5, occludin, ZO-1 or ZO-2 or the adherens junction-associated proteins beta-catenin or p120cas did not change. Thus, opening of BBB TJs is not readily accompanied by the complete loss of the junctional localization of TJ proteins.

Animals↗
PubMed1989

Polarized expression of an apical membrane glycoprotein is established before functional tight junctions have developed in MCF-7 cells.

A milk-fat globule membrane antigen, designated MAM-6 and detected immunocytochemically by the monoclonal antibody 115D8, is expressed apically in confluent MCF-7 monolayer cultures. Immediately after preparation of a single-cell suspension, MAM-6 appears on the entire cell surface. However, polarized apical expression of MAM-6 is restored as early as 2-6 hr after plating of unpolarized cells, before functional tight junctions are established, as judged by freeze-fracture and ruthenium red permeability. Quantitative immunogold cytochemistry reveals that the apical:basal ratio of MAM-6 expression was about 17:1 after 6 hr. Tight junctions developed as late as 12-24 hr after plating. At this time the apical:basal MAM-6 ratio was about 30:1 (as compared to about 50:1 in control monolayers).

Antigens, Neoplasm↗
PubMed2003

Infection of human and bovine epithelial cells with Cryptosporidium andersoni induces apoptosis and disrupts tight junctional ZO-1: effects of epidermal growth factor.

The effects of Cryptosporidium andersoni on human or bovine epithelia are poorly defined. Epidermal growth factor inhibits colonisation of the gastrointestinal epithelium with bacteria and the enteric protozoan parasite Giardia lamblia. This study characterised whether C. andersoni infects human or bovine epithelial cells in vitro, assessed its impact on apoptosis and tight junctional Zonula-Occludens-1, and determined whether these effects may be altered by epidermal growth factor. Monolayers of human colonic CaCo(2) cells, SCBN (non-malignant small intestinal epithelial cells), and Madin Darby bovine kidney epithelial cell lines (MDBK and NBL-1) were grown to confluency in Dulbecco's Modified Eagle Medium. Monolayers were assigned to one of three experimental groups-(1) control: exposed to culture medium alone; (2) untreated: exposed to 10(3) live C. andersoni oocysts or (3) epidermal growth factor-treated: apically pre-treated with recombinant human epidermal growth factor and then exposed to Cryptosporidium. Oocyst viability, infection with Cryptosporidium, apoptosis, and integrity of tight junctional Zonula-Occludens-1 were assessed. In addition, live Cryptosporidium oocysts were incubated with epidermal growth factor to assess whether epidermal growth factor had cryptosporicidial activity. Cryptosporidium andersoni oocysts infected all human and bovine monolayers, increased nuclear fragmentation, and disrupted Zonula-Occludens-1. Apical epidermal growth factor significantly reduced infection with C. andersoni in all cell lines and inhibited the Cryptosporidium-induced apoptosis and disruption of Zonula-Occludens-1. Epidermal growth factor did not affect oocyst viability.

Animals↗
PubMed1989

The effect of gentamicin-induced hair cell loss on the tight junctions of the reticular lamina.

Freeze fracture has been used to examine junctional complexes within the gentamicin damaged guinea pig organ of Corti during the period of structural reorganisation associated with hair cell loss. When examined up to 28 days after a regime of chronic gentamicin administration, no significant alterations to the pattern or nature of the complex tight junctions associated with the hair cell apex could be detected. In contrast, marked abnormalities of the junctions between adjacent supporting cells, and evidence of formation of new gap junctions on the lateral membranes of Deiter's cell was seen. The use of lanthanum as an electron dense tracer in thin sections also showed that no breakdown of permeability barriers occurred in the reticular lamina. These results support the concept of a controlled response by the organ of Corti, with conservation of the tight junction of the hair cell and maintenance of permeability barriers. Early changes to the membranes of the stereocilia and lateral cisternae of the outer hair cells were also identified. These may indicate direct effects of gentamicin upon membrane structure.

Animals↗
PubMed2005

Characterisation of adherens and tight junctional molecules in normal animal larynx; determining a suitable model for studying molecular abnormalities in human laryngopharyngeal reflux.

BACKGROUND: The disruption of intercellular junctions in the larynx is a pathological feature of laryngopharyngeal reflux (LPR). Good experimental models are necessary to gain greater insight into the molecular mechanisms and alterations that result from abnormal exposure of the laryngeal epithelium to acid refluxate. AIMS: To characterise laryngeal tissues from different species to determine the most suitable for use in experimental studies of LPR. METHODS: Human and non-human laryngeal tissues (mouse, rat, guinea pig, porcine, and rabbit) were studied. Histological characterisation was performed by light microscopy. The expression and subcellular localisation of adherens junctional molecules (E-cadherin and beta catenin) was evaluated by immunohistochemistry, and tight junction molecules (occludin and zonula occludens 1 (ZO-1)) by western blotting. The ultrastructural features of porcine and human tissue were assessed by electron microscopy. RESULTS: Porcine tissue revealed both respiratory-type and stratified squamous epithelium, as seen in the human larynx. The expression and subcellular localisation of the E-cadherin-catenin complex was detected in all species except mouse and rat. The pattern of ZO-1 and occludin expression was preserved in all species. CONCLUSION: The expression of intercellular junctional complexes in porcine epithelium is similar to that seen in humans. These results confirm the suitability of these species to study molecular mechanisms of LPR in an experimental system.

Adherens Junctions↗
PubMed1999

Role of tyrosine phosphorylation in the reassembly of occludin and other tight junction proteins.

After the simulation of anoxia by ATP depletion of MDCK cell monolayers with metabolic inhibitors, the tight junction (TJ) is known to become structurally perturbed, leading to loss of the permeability barrier. Peripheral TJ proteins such as zonula occludens 1 (ZO-1), ZO-2, and cingulin become extremely insoluble and associate into large macromolecular complexes (T. Tsukamoto and S. K. Nigam. J. Biol. Chem. 272: 16133-16139, 1997). For up to 3 h, this process is reversible by ATP repletion. We now show that the reassembly process depends on tyrosine phosphorylation. Recovery of transepithelial electrical resistance in ATP-replete monolayers was markedly inhibited by the tyrosine kinase inhibitor, genistein. Indirect immunofluorescence revealed a decrease in staining of occludin, a membrane component of the TJ, in the region of the TJ after ATP depletion, which reversed after ATP repletion; this reversal process was inhibited by genistein. Examination of the Triton X-100 solubilities of occludin and several nonmembrane TJ proteins revealed a shift of occludin and nonmembrane TJ proteins into an insoluble pool following ATP depletion. These changes reversed after ATP repletion, and the movement of insoluble occludin, ZO-1, and ZO-2 back into the soluble pool was again via a genistein-sensitive mechanism. Rate-zonal centrifugation analyses of detergent-soluble TJ proteins showed a reversible increase in higher density fractions following ATP depletion-repletion, although this change was not affected by genistein. In 32P-labeled cells, dephosphorylation of all studied TJ proteins was observed during ATP depletion, followed by rephosphorylation during ATP repletion; rephosphorylation of occludin was inhibited by genistein. Furthermore, during the ATP repletion phase, tyrosine phosphorylation of Triton X-100-insoluble occludin, which is localized at the junction, as well as ZO-2, p130/ZO-3 (though not ZO-1), and other proteins was evident; this tyrosine phosphorylation was completely inhibited by genistein. This indicates that tyrosine kinase activity is necessary for TJ reassembly during ATP repletion and suggests an important role for the tyrosine phosphorylation of occludin, ZO-2, p130/ZO-3, and possibly other proteins in the processes involved in TJ (re)formation.

Adenosine Triphosphate↗
PubMed2000

Clostridium perfringens enterotoxin binds to the second extracellular loop of claudin-3, a tight junction integral membrane protein.

Claudins (claudin-1 to -18) with four transmembrane domains and two extracellular loops constitute tight junction strands. The peptide toxin Clostridium perfringens enterotoxin (CPE) has been shown to bind to claudin-3 and -4, but not to claudin-1 or -2. We constructed claudin-1/claudin-3 chimeric molecules and found that the second extracellular loop of claudin-3 conferred CPE sensitivity on L fibroblasts. Furthermore, overlay analyses revealed that the second extracellular loop of claudin-3 specifically bound to CPE at the K(a) value of 1.0x10(8) M(-1). We concluded that the second extracellular loop is the site through which claudin-3 interacts with CPE on the cell surface.

Animals↗
PubMed1990

Rabbit retinal Müller cells in cell culture show gap and tight junctions which they do not express in situ.

Retinae of early postnatal rabbits were enzymatically dissociated and explanted in a culture system. The prospective myelinated region was discarded in order to avoid the presence of astrocytic or mesenchymal cells. After about 14 days in vitro (DIV), outgrowing glial (Müller) cells formed what light optically appeared to be confluent monolayers but by electron microscopy was shown to consist of flat epithelioid cells which overlapped considerably by extension of cytoplasmic tongues. Applying the freeze-fracture technique, apposed membranes of these cells were demonstrated to express infrequently but consistently both gap and tight junctions. This kind of junctions has never been observed on the membrane of rabbit Müller cells in situ. In comparison with Müller cell membranes in situ, the density of intramembrane particles was considerably reduced. Orthogonal arrays of particles which are characteristic elements of Müller cells in situ were not detected. Our results suggest that in homogeneous cell culture, Müller cells form some kind of epithelium-like specialized intercellular junctions. This situation resembles that of closely related glial cell types which form homogeneous layers in situ as e.g. retinal pigment epithelium cells expressing tight junctions, and marginal astrocytes being coupled by extensive gap junctions.

Animals↗
PubMed2005

Advances in nasal drug delivery through tight junction technology.

New approaches for enhancing intranasal drug delivery based on recent discoveries on the molecular biology of tight junctions (TJ) are significantly improving the bioavailability of 'non-Lipinsky' small molecules, and peptide, protein and oligonucleotide drugs. As knowledge of the structure and function of the TJ has developed, so has the ability to identify mechanism-based TJ modulators using high-throughput molecular biology-based screening methods. The present review focuses on recent developments on the TJ protein complex as a lipid raft-like membrane microdomain, the emerging role of unique endocytic pathways in regulating TJ dynamics, and the utility of techniques such as RNA interference and phage display to study TJ components and identify novel peptides and related molecules that can modulate their function. Experimental and statistical methodologies used for the identification of new classes of TJ modulators are described, which are capable of reversibly opening TJ barriers with broad potential to significantly improve intranasal and, eventually, oral drug delivery. The development of an advanced intranasal formulation for the obesity therapeutic PYY(3-36), the endogenous Y2 receptor agonist is also reviewed.

Administration, Intranasal↗
PubMed1999

Claudin multigene family encoding four-transmembrane domain protein components of tight junction strands.

Two related integral membrane proteins, claudin-1 and -2, recently were identified as novel components of tight junction (TJ) strands. Here, we report six more claudin-like proteins, indicating the existence of a claudin gene family. Three of these were reported previously as RVP1, Clostridium perfringens enterotoxin receptor, and TMVCF, but their physiological functions were not determined. Through similarity searches followed by PCR, we isolated full length cDNAs of mouse RVP1, Clostridium perfringens enterotoxin receptor, and TMVCF as well as three mouse claudin-like proteins and designated them as claudin-3 to -8, respectively. All of these claudin family members showed similar patterns on hydrophilicity plots, which predicted four transmembrane domains in each molecule. Northern blotting showed that the tissue distribution pattern varied significantly, depending on claudin species. Similarly to claudin-1 and -2, when these claudins were HA-tagged and introduced into cultured Madin-Darby canine kidney cells, all showed a tendency to concentrate at TJs. Immunofluorescence and immunoelectron microscopy with polyclonal antibodies specific for claudin-3, -4, or -8 revealed that these molecules were exclusively concentrated at TJs in the liver and/or kidney. These findings indicated that multiple claudin family members are involved in the formation of TJ strands in various tissues.

Amino Acid Sequence↗
PubMed2004

The C-terminal cytoplasmic tail of claudins 1 and 5 but not its PDZ-binding motif is required for apical localization at epithelial and endothelial tight junctions.

Claudins are a family of tetraspan transmembrane proteins that represent the major constituents of epithelial and endothelial tight junctions (TJs). They form TJ strands representing the major barrier regulating paracellular transport of solutes and water. Intracellularly, claudins are connected via a C-terminal PDZ-binding motif with several TJ-associated proteins containing PDZ domains. Although these interactions can provide a link to the actin cytoskeleton, they appear to be dispensable for the TJ localization of claudins. To identify TJ-targeting elements in the C-terminal cytoplasmic domains of the claudins 1 and 5, we generated a series of C-terminal deletion mutants and analyzed their distribution in polarized epithelial (MDCK) and endothelial (HMEC-1) cells. TJ localization was revealed by establishing an in vivo cross-linking approach that stabilized claudin-TJ interactions. We show that residues located C-terminal to the last transmembrane domain are required for the proper targeting to apical TJ.s. While claudin derivatives lacking only the very C-terminal PDZ-binding motif continue to localize to TJs, mutants lacking the entire C-terminal juxtamembrane sequence do not associate with TJs and accumulate in intracellular structures. This indicates that crucial determinants for stable TJ incorporation of claudins reside in a cytoplasmic C-terminal sequence which up to now has not been implicated in specific protein-protein interactions.

Amino Acid Motifs↗
PubMed1999

Molecular characterization of the tight junction protein ZO-1 in MDCK cells.

Most of the information on the structure and function of the tight junction (TJ) has been obtained in MDCK cells. Accordingly, we have sequenced ZO-1 in this cell type, because this protein is involved in the response of the TJ to changes in Ca2+, phosphorylation, and the cytoskeleton. ZO-1 of MDCK cells comprises 6805 bp with a predicted open reading frame of 1769 amino acids. This sequence is 92 and 87% homologous to human and mouse ZO-1, respectively. Two nuclear sorting signals located at the PDZ1 and GK domains and 17 SH3 putative binding sites at the proline-rich domain were detected. We found two new splicing regions at the proline-rich region: beta had not been reported in human and mouse counterparts, and gamma, which was previously sequenced in human and mouse ZO-1, is now identified as a splicing region. The expression of different beta and gamma isoforms varies according to the tissue tested. With the information provided by the sequence, Southern blot, and PCR experiments we can predict a single genomic copy of MDCK-ZO-1 that is at least 13.16 kb long. MDCK-ZO-1 mRNA is 7.4 kb long. Its expression is regulated by calcium, while the expression of MDCK-ZO-1 protein is not.

Alternative Splicing↗
PubMed1980

Tight junctions in the ependyma of the spinal cord of the urodele Pleurodeles waltlii.

The ependymal junction pattern in the spinal cord of postmetamorphic ribbed newts has been studied, using transmission electron microscopy of ultrathin sections of normal animals and of animals perfused through the IVth ventricle with lanthanum. Contrary to what has been observed in mammalian CNS, the ependyma of the urodelan spinal cord is furnished with tight junctions that seal the luminal border of the terminal bars. These occludens junctions are made up of two to seven punctate fusions of the plasma membranes. Lanthanum tracer remains restricted inside the lumen of the central canal, being stopped at the first punctate fusion on its way through the intercellular clefts. Beyond this point, the extracellular space contains no tracer material. Besides tight junctions, intermediate, desmosomal and gap junctions are also present. Gap junctions and desmosomes are not present in CSF-contacting neurons. It is suggested that ependyma with occluding junctions (special ependyma) overlay the regions of the CNS where the ependymal cells significantly modify the composition of both intercellular and cerebrospinal fluids, through secretory, transporting and permeability control activities.

Animals↗