Ischemic preconditioning enhances integrity of coronary endothelial tight junctions

Ischemic preconditioning (IPC) is one of the most effective procedures known to protect hearts against ischemia/reperfusion (IR) injury. Tight junction (TJ) barriers occur between coronary endothelial cells. TJs provide barrier function to maintain the homeostasis of the inner environment of tissues. However, the effect of IPC on the structure and function of cardiac TJs remains unknown. We tested the hypothesis that myocardial IR injury ruptures the structure of TJs and impairs endothelial permeability whereas IPC preserves the structural and functional integrity of TJs in the blood-heart barrier. Langendorff hearts from C57BL/6J mice were prepared and perfused with Krebs-Henseleit buffer. Cardiac function, creatine kinase release, and myocardial edema were measured. Cardiac TJ function was evaluated by measuring Evans blue-conjugated albumin (EBA) content in the extravascular compartment of hearts. Expression and translocation of zonula occludens (ZO)-2 in IR and IPC hearts were detected with Western blot. A subset of hearts was processed for the observation of ultra-structure of cardiac TJs with transmission electron microscopy. There were clear TJs between coronary endothelial cells of mouse hearts. IR caused the collapse of TJs whereas IPC sustained the structure of TJs. IR increased extravascular EBA content in the heart and myocardial edema but decreased the expression of ZO-2 in the cytoskeleton. IPC maintained the structure of TJs. Cardiac EBA content and edema were reduced in IPC hearts. IPC enhanced the translocation of ZO-2 from cytosol to cytoskeleton. In conclusion, TJs occur in normal mouse heart. IPC preserves the integrity of TJ structure and function that are vulnerable to IR injury.     

Crosstalk of tight junction components with signaling pathways

Tight junctions (TJs) regulate the passage of ions and molecules through the paracellular pathway in epithelial and endothelial cells. TJs are highly dynamic structures whose degree of sealing varies according to external stimuli, physiological and pathological conditions. In this review we analyze how the crosstalk of protein kinase C, protein kinase A, myosin light chain kinase, mitogen-activated protein kinases, phosphoinositide 3-kinase and Rho signaling pathways is involved in TJ regulation triggered by diverse stimuli. We also report how the phosphorylation of the main TJ components, claudins, occludin and ZO proteins, impacts epithelial and endothelial cell function.     

Crosstalk of tight junction components with signaling pathways

Tight junctions (TJs) regulate the passage of ions and molecules through the paracellular pathway in epithelial and endothelial cells. TJs are highly dynamic structures whose degree of sealing varies according to external stimuli, physiological and pathological conditions. In this review we analyze how the crosstalk of protein kinase C, protein kinase A, myosin light chain kinase, mitogen-activated protein kinases, phosphoinositide 3-kinase and Rho signaling pathways is involved in TJ regulation triggered by diverse stimuli. We also report how the phosphorylation of the main TJ components, claudins, occludin and ZO proteins, impacts epithelial and endothelial cell function.     

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.     

Proinflammatory cytokine-induced tight junction remodeling through dynamic self-assembly of claudins

Tight junctions (TJs) are dynamic, multiprotein intercellular adhesive contacts that provide a vital barrier function in epithelial tissues. TJs are remodeled during physiological development and pathological mucosal inflammation, and differential expression of the claudin family of TJ proteins determines epithelial barrier properties. However, the molecular mechanisms involved in TJ remodeling are incompletely understood. Using acGFP-claudin 4 as a biosensor of TJ remodeling, we observed increased claudin 4 fluorescence recovery after photobleaching (FRAP) dynamics in response to inflammatory cytokines. Interferon γ and tumor necrosis factor α increased the proportion of mobile claudin 4 in the TJ. Up-regulation of claudin 4 protein rescued these mobility defects and cytokine-induced barrier compromise. Furthermore, claudins 2 and 4 have reciprocal effects on epithelial barrier function, exhibit differential FRAP dynamics, and compete for residency within the TJ. These findings establish a model of TJs as self-assembling systems that undergo remodeling in response to proinflammatory cytokines through a mechanism of heterotypic claudin-binding incompatibility.     

Endothelial tight junctions and their regulatory signaling pathways in vascular homeostasis and disease

Endothelial tight junctions (TJs) regulate the transport of water, ions, and molecules through the paracellular pathway, serving as an important barrier in blood vessels and maintaining vascular homeostasis. In endothelial cells (ECs), TJs are highly dynamic structures that respond to multiple external stimuli and pathological conditions. Alterations in the expression, distribution, and structure of endothelial TJs may lead to many related vascular diseases and pathologies. In this review, we provide an overview of the assessment methods used to evaluate endothelial TJ barrier function both in vitro and in vivo and describe the composition of endothelial TJs in diverse vascular systems and ECs. More importantly, the direct phosphorylation and dephosphorylation of TJ proteins by intracellular kinases and phosphatases, as well as the signaling pathways involved in the regulation of TJs, including and the protein kinase C (PKC), PKA, PKG, Ras homolog gene family member A (RhoA), mitogen-activated protein kinase (MAPK), phosphatidylinositol 3-kinase (PI3K)/Akt, and Wnt/β-catenin pathways, are discussed. With great advances in this area, targeting endothelial TJs may provide novel treatment for TJ-related vascular pathologies.     

Assembly of epithelial tight junctions is negatively regulated by Par6

Epithelial cells display apical-basal polarity, and the apical surface is segregated from the basolateral membranes by a barrier called the tight junction (TJ). TJs are constructed from transmembrane proteins that form cell-cell contacts-claudins, occludin, and junctional adhesion molecule (JAM)-plus peripheral proteins such as ZO-1. The Par proteins (partitioning-defective) Par3 and Par6, plus atypical protein kinase C (aPKC) function in the formation or maintenance of TJs and more generally in metazoan cell polarity establishment. Par6 contains a PDZ domain and a partial CRIB (Cdc42/Rac interactive binding) domain and binds the small GTPase Cdc42. Here, we show that Par6 inhibits TJ assembly in MDCK II epithelial cells after their disruption by Ca(2+) depletion but does not inhibit adherens junction (AJ) formation. Transepithelial resistance and paracellular diffusion assays confirmed that assembly of functional TJs is delayed by Par6 overexpression. Strikingly, the isolated, N-terminal fragment of PKCzeta, which binds Par6, also inhibits TJ assembly. Activated Cdc42 can disrupt TJs, but neither a dominant-negative Cdc42 mutant nor the CRIB domain of gammaPAK (p21-activated kinase), which inhibits Cdc42 function, observably inhibit TJ formation. These results suggest that Cdc42 and Par6 negatively regulate TJ assembly in mammalian epithelial cells.     

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.     

Endocytosis of epithelial apical junctional proteins by a clathrin-mediated pathway into a unique storage compartment

The adherens junction (AJ) and tight junction (TJ) are key regulators of epithelial polarity and barrier function. Loss of epithelial phenotype is accompanied by endocytosis of AJs and TJs via unknown mechanisms. Using a model of calcium depletion, we defined the pathway of internalization of AJ and TJ proteins (E-cadherin, p120 and beta-catenins, occludin, JAM-1, claudins 1 and 4, and ZO-1) in T84 epithelial cells. Proteinase protection assay and immunocytochemistry revealed orchestrated internalization of AJs and TJs into a subapical cytoplasmic compartment. Disruption of caveolae/lipid rafts did not prevent endocytosis, nor did caveolin-1 colocalize with internalized junctional proteins. Furthermore, AJ and TJ proteins did not colocalize with the macropinocytosis marker dextran. Inhibitors of clathrin-mediated endocytosis blocked internalization of AJs and TJs, and junctional proteins colocalized with clathrin and alpha-adaptin. AJ and TJ proteins were observed to enter early endosomes followed by movement to organelles that stained with syntaxin-4 but not with markers of late and recycling endosomes, lysosomes, or Golgi. These results indicate that endocytosis of junctional proteins is a clathrin-mediated process leading into a unique storage compartment. Such mechanisms may mediate the disruption of intercellular contacts during normal tissue remodeling and in pathology.     

MgcRacGAP interacts with cingulin and paracingulin to regulate Rac1 activation and development of the tight junction barrier during epithelial junction assembly

The regulation of Rho-family GTPases is crucial to direct the formation of cell–cell junctions and tissue barriers. Cingulin (CGN) and paracingulin (CGNL1) control RhoA activation in epithelial cells by interacting with RhoA guanidine exchange factors. CGNL1 depletion also inhibits Rac1 activation during junction assembly. Here we show that, unexpectedly, Madin–Darby canine kidney epithelial cells depleted of both CGN and CGNL1 (double-KD cells) display normal Rac1 activation and tight junction (TJ) formation, despite decreased junctional recruitment of the Rac1 activator Tiam1. The expression of the Rac1 inhibitor MgcRacGAP is decreased in double-KD cells, and the barrier development and Rac1 activation phenotypes are rescued by exogenous expression of MgcRacGAP. MgcRacGAP colocalizes with CGN and CGNL1 at TJs and forms a complex and interacts directly in vitro with CGN and CGNL1. Depletion of either CGN or CGNL1 in epithelial cells results in decreased junctional localization of MgcRacGAP but not of ECT2, a centralspindlin-interacting Rho GEF. These results provide new insight into coordination of Rho-family GTPase activities at junctions, since apical accumulation of CGN and CGNL1 at TJs during junction maturation provides a mechanism to spatially restrict down-regulation of Rac1 activation through the recruitment of MgcRacGAP.     

Nonredundant roles of cytoplasmic β- and γ-actin isoforms in regulation of epithelial apical junctions

Association with the actin cytoskeleton is critical for normal architecture and dynamics of epithelial tight junctions (TJs) and adherens junctions (AJs). Epithelial cells express β-cytoplasmic (β-CYA) and γ-cytoplasmic (γ-CYA) actins, which have different cellular localization and functions. This study elucidates the roles of cytoplasmic actins in regulating structure and remodeling of AJs and TJs in model intestinal epithelia. Immunofluorescence labeling and latrunculin B treatment reveal affiliation of dynamic β-CYA filaments with newly assembled and mature AJs, whereas an apical γ-CYA pool is composed of stable perijunctional bundles and rapidly turning-over nonjunctional filaments. The functional effects of cytoplasmic actins on epithelial junctions are examined by using isoform-specific small interfering RNAs and cell-permeable inhibitory peptides. These experiments demonstrate unique roles of β-CYA and γ-CYA in regulating the steady-state integrity of AJs and TJs, respectively. Furthermore, β-CYA is selectively involved in establishment of apicobasal cell polarity. Both actin isoforms are essential for normal barrier function of epithelial monolayers, rapid AJ/TJ reassembly, and formation of three-dimensional cysts. Cytoplasmic actin isoforms play unique roles in regulating structure and permeability of epithelial junctions.     

Vascular Endothelial Tight Junctions and Barrier Function Are Disrupted by 15(S)-Hydroxyeicosatetraenoic Acid Partly via Protein Kinase C?-mediated Zona Occludens-1 Phosphorylation at Threonine 770/772

Disruption of tight junctions (TJs) perturbs endothelial barrier function and promotes inflammation. Previously, we have shown that 15(S)-hydroxyeicosatetraenoic acid (15(S)-HETE), the major 15-lipoxygenase 1 (15-LO1) metabolite of arachidonic acid, by stimulating zona occludens (ZO)-2 tyrosine phosphorylation and its dissociation from claudins 1/5, induces endothelial TJ disruption and its barrier dysfunction. Here, we have studied the role of serine/threonine phosphorylation of TJ proteins in 15(S)-HETE-induced endothelial TJ disruption and its barrier dysfunction. We found that 15(S)-HETE enhances ZO-1 phosphorylation at Thr-770/772 residues via PKCϵ-mediated MEK1-ERK1/2 activation, causing ZO-1 dissociation from occludin, disrupting endothelial TJs and its barrier function, and promoting monocyte transmigration; these effects were reversed by T770A/T772A mutations. In the arteries of WT mice ex vivo, 15(S)-HETE also induced ZO-1 phosphorylation and endothelial TJ disruption in a PKCϵ and MEK1-ERK1/2-dependent manner. In line with these observations, in WT mice high fat diet feeding induced 12/15-lipoxygenase (12/15-LO) expression in the endothelium and caused disruption of its TJs and barrier function. However, in 12/15-LO^−/− mice, high fat diet feeding did not cause disruption of endothelial TJs and barrier function. These observations suggest that the 12/15-LO-12/15(S)-HETE axis, in addition to tyrosine phosphorylation of ZO-2, also stimulates threonine phosphorylation of ZO-1 in the mediation of endothelial TJ disruption and its barrier dysfunction.     

The role of epithelial tight junctions involved in pathogen infections

Tight junctions (TJs) are sealing complexes between adjacent epithelial cells, functioning by controlling paracellular passage and maintaining cell polarity. These functions of TJs are primarily based on structural integrity as well as dynamic regulatory balance, indicating plasticity of TJ in response to external stimuli. An indispensable role of TJs involved in pathogen infection has been widely demonstrated since disruption of TJs leads to a distinct increase in paracellular permeability and polarity defects which facilitate viral or bacterial entry and spread. In addition to pathological changes in TJ integrity, TJ proteins such as occludin and claudins can either function as receptors for pathogen entry or interact with viral/bacterial effector molecules as an essential step for characterizing an infective stage. This suggests a more complicated role for TJ itself and especially specific TJ components. Thus, this review surveys the role of the epithelial TJs involved in various pathogen infections, and extends TJ targeted therapeutic and pharmacological application prospects.     

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.     

Peptidase allergen Der p 1 initiates apoptosis of epithelial cells independently of tight junction proteolysis

Loss of epithelial cell polarity, which can arise following disruption of tight junctions (TJs), is a precursor to the care-fully orchestrated removal of moribund cells from epithelia in apoptosis. Ordinarily, this cycle of events has minimally disruptive effects on the function of the epithelial barrier, but some agents have been identified that induce apoptosis and promote epithelial leakiness. The allergen Der p 1 is a cysteine peptidase that cleaves TJ adhesion proteins and induces apoptosis in epithelial cells. This suggests the possibility that, at least for some inducers of apoptosis, these events might be causally linked. We report here that Der p 1 induces epithelial apoptosis before outright cell detachment and that apoptosis occurs within the same time span as increased paracellular permeability in polarized epithelial monolayers. Whilst TJ-deficient BEAS-2B cells were resistant to Der p 1-induced apoptosis, the cell line 1HAEo-, which was also TJ deficient, was sensitive to Der p 1, providing evidence against TJ proteolysis as a cause of apoptosis. To provide direct evidence, we propagated cells that normally express TJs in low calcium medium that prevented intercellular junction assembly. These cells retained full susceptibility to Der p 1, indicating that Der p 1-induced apoptosis is independent from TJ proteolysis.     

Peptidase allergen Der p 1 initiates apoptosis of epithelial cells independently of tight junction proteolysis

Loss of epithelial cell polarity, which can arise following disruption of tight junctions (TJs), is a precursor to the carefully orchestrated removal of moribund cells from epithelia in apoptosis. Ordinarily, this cycle of events has minimally disruptive effects on the function of the epithelial barrier, but some agents have been identified that induce apoptosis and promote epithelial leakiness. The allergen Der p 1 is a cysteine peptidase that cleaves TJ adhesion proteins and induces apoptosis in epithelial cells. This suggests the possibility that, at least for some inducers of apoptosis, these events might be causally linked. We report here that Der p 1 induces epithelial apoptosis before outright cell detachment and that apoptosis occurs within the same time span as increased paracellular permeability in polarized epithelial monolayers. Whilst TJ-deficient BEAS-2B cells were resistant to Der p 1-induced apoptosis, the cell line 1HAEo-, which was also TJ deficient, was sensitive to Der p 1, providing evidence against TJ proteolysis as a cause of apoptosis. To provide direct evidence, we propagated cells that normally express TJs in low calcium medium that prevented intercellular junction assembly. These cells retained full susceptibility to Der p 1, indicating that Der p 1-induced apoptosis is independent from TJ proteolysis.     

Occludin-deficient Embryonic Stem Cells Can Differentiate into Polarized Epithelial Cells Bearing Tight Junctions

Occludin is the only known integral membrane protein of tight junctions (TJs), and is now believed to be directly involved in the barrier and fence functions of TJs. Occludin-deficient embryonic stem (ES) cells were generated by targeted disruption of both alleles of the occludin gene. When these cells were subjected to suspension culture, they aggregated to form simple, and then cystic embryoid bodies (EBs) with the same time course as EB formation from wild-type ES cells. Immunofluorescence microscopy and ultrathin section electron microscopy revealed that polarized epithelial (visceral endoderm-like) cells were differentiated to delineate EBs not only from wild-type but also from occludin-deficient ES cells. Freeze fracture analyses indicated no significant differences in number or morphology of TJ strands between wild-type and occludin-deficient epithelial cells. Furthermore, zonula occludens (ZO)-1, a TJ-associated peripheral membrane protein, was still exclusively concentrated at TJ in occludin-deficient epithelial cells. In good agreement with these morphological observations, TJ in occludin-deficient epithelial cells functioned as a primary barrier to the diffusion of a low molecular mass tracer through the paracellular pathway. These findings indicate that there are as yet unidentified TJ integral membrane protein(s) which can form strand structures, recruit ZO-1, and function as a barrier without occludin.     

Retinoid X receptor alpha and retinoic acid receptor gamma mediate expression of genes encoding tight-junction proteins and barrier function in F9 cells during visceral endodermal differentiation

Retinoids are critical for differentiation of columnar epithelial cells and for preventing metaplasia of these cells into stratified squamous epithelial cells, in which tight junctions (TJs) are essentially absent. This implies that retinoids might play important roles in regulating the structures and functions of TJs of columnar epithelium. F9 murine embryonal carcinoma cells differentiate into epithelial cells resembling visceral endoderm bearing TJs, when grown in suspension as aggregates in the presence of retinoic acid (RA). We show that RA induces the TJ structure and expression of several TJ-associated molecules, such as ZO-1, occludin, claudin-6, and claudin-7, as well as a barrier function in the genetically engineered cell line F9:rtTA:Cre-ER(T) L32T2, which allows sophisticated genetic manipulations simply by addition of ligands (H. Chiba et al., 2000, Exp. Cell Res. 260, 334-339). Interestingly, our data indicate that a barrier for small substances is generated after that for large ones during de novo formation of TJs. We also compared the RA-induced expression of TJ components and barrier function in RXRalpha(-/-)-RARgamma(-/-) F9 cells with those in wild-type cells and show that the retinoid signals for transduction of these events are mediated by specific RXR-RAR pairs.     

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.