VEGF increases BMEC monolayer permeability by affecting occludin expression and tight junction assembly

Tight junctions between brain microvessel endothelial cells (BMECs) maintain the blood-brain barrier. Barrier breakdown is associated with brain tumors and central nervous system diseases. Tumor cell-secreted vascular endothelial growth factor (VEGF) increases microvasculature permeability in vivo and is correlated with the induction of clinically severe brain tumor edema. Here we investigated the permeability-increasing effect and tight junction formation of VEGF. By measuring [(14)C]sucrose flux and transendothelial electrical resistance (TER) across BMEC monolayer cultures, we found that VEGF increased sucrose permeability and decreased TER. VEGF also caused a loss of occludin and ZO-1 from the endothelial cell junctions and changed the staining pattern of the cell boundary. Western blot analysis of BMEC lysates revealed that the level of occludin but not of ZO-1 was lowered by VEGF treatment. These results suggest that VEGF increases BMEC monolayer permeability by reducing occludin expression and disrupting ZO-1 and occludin organization, which leads to tight junction disassembly. Occludin and ZO-1 appear to be downstream effectors of the VEGF signaling pathway.     

Occludin regulates actin cytoskeleton in endothelial cells

Occludin is a major membrane component of tight junctions of endothelial cells, though the role of this molecule is not fully understood. RLE cells, derived from rat lung endothelial cells, express a negligible level of occludin with clear expression of E-cadherin and ZO-1 at cell junctions. Introduction of occludin by transfection induced clear junctional expression of occludin with few or no changes of expression of E-cadherin and ZO-1. The paracellular barrier function, as determined by transelectrical resistance and flux of non-ionic small molecules, was not detectably upregulated. When cells expressing occludin were cocultured with RLE cells null for occludin, clear junctional expression of occludin was observed irrespective of the expression of occludin on the apposing cells. Cortical actin was developed at the site of these occludin positive cell junctions. Treatment of cells with an actin depolymerizing agent, mycalolide B, abolished junctional expression of occludin together with E-cadherin and circumferential actin. ZO-1 showed relative resistance to this actin depolymerizing treatment and was maintained at the cell junctions, though fragmentation of immunoreactivity was detectable. Collectively, junctional expression of occludin was not associated with paracellular barrier function in this cell line. There was, however, a close correlation of occludin with the actin cytoskeleton, indicating a role of occludin as an important molecule in the regulation of the actin cytoskeleton in endothelial cells.     

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.     

Correlation of tight junction morphology with the expression of tight junction proteins in blood-brain barrier endothelial cells

Endothelial cells of the blood-brain barrier form complex tight junctions, which are more frequently associated with the protoplasmic (P-face) than with the exocytoplasmic (E-face) membrane leaflet. The association of tight junctional particles with either membrane leaflet is a result of the expression of various claudins, which are transmembrane constituents of tight junction strands. Mammalian brain endothelial tight junctions exhibit an almost balanced distribution of particles and lose this morphology and barrier function in vitro. Since it was shown that the brain endothelial tight junctions of submammalian species form P-face-associated tight junctions of the epithelial type, the question of which molecular composition underlies the morphological differences and how do these brain endothelial cells behave in vitro arose. Therefore, rat and chicken brain endothelial cells were investigated for the expression of junctional proteins in vivo and in vitro and for the morphology of the tight junctions. In order to visualize morphological differences, the complexity and the P-face association of tight junctions were quantified. Rat and chicken brain endothelial cells form tight junctions which are positive for claudin-1, claudin-5, occludin and ZO-1. In agreement with the higher P-face association of tight junctions in vivo, chicken brain endothelia exhibited a slightly stronger labeling for claudin-1 at membrane contacts. Brain endothelial cells of both species showed a significant alteration of tight junctions in vitro, indicating a loss of barrier function. Rat endothelial cells showed a characteristic switch of tight junction particles from the P-face to the E-face, accompanied by the loss of claudin-1 in immunofluorescence labeling. In contrast, chicken brain endothelial cells did not show such a switch of particles, although they also lost claudin-1 in culture. These results demonstrate that the maintenance of rat and chicken endothelial barrier function depends on the brain microenvironment. Interestingly, the alteration of tight junctions is different in rat and chicken. This implies that the rat and chicken brain endothelial tight junctions are regulated differently.     

Apoptosis in mouse amniotic epithelium is induced by activated macrophages through the TNF receptor type 1/TNF pathway

The amniotic epithelium is in direct contact with the amniotic fluid and has tight junctions. The amniotic tight junctions function as a barrier to restrict fluid flux via the amniotic membrane during midpregnancy in the mouse. However, during late pregnancy, amniotic fluid volume significantly decreases in association with the disruption of amniotic tight junctions. The disruption of amniotic tight junctions is caused by apoptosis in the amniotic epithelium on Embryonic Day 17 (E17). In this study, we examine the molecular mechanisms underlying apoptosis of the amniotic epithelium of the mouse. We found that from E16, the number of activated macrophages that express high levels of NOS2 and tumor necrosis factor (TNF) increase in amniotic fluid. TNF receptor type 1 (TNFR1) was detectable from E16 onward. On E17, amniotic epithelial cells expressing TNFR1 became TUNEL positive, suggesting that TNF/TNFR1 signaling may initiate apoptosis. To further confirm the role of TNF/TNFR1 signaling, WP9QY, a TNFR1 antagonist, was injected into the amniotic cavity and was found to significantly reduce the numbers of apoptotic cells in the E17 amniotic epithelium. Furthermore, dehydroxymethylepoxyquinomicin, a specific nuclear factor-kappa B inhibitor, was found to inhibit TNF production in macrophages and amniotic apoptosis in vivo. Finally, we showed that injection of TNF into the amniotic cavity induces early onset of apoptosis. These results indicate that amniotic apoptosis is induced by the TNF pathway via TNFR1 expressed in the amniotic epithelial cells and that activation of macrophages may trigger amniotic apoptosis.     

Recombinant Human Sonic Hedgehog Protein Regulates the Expression of ZO-1 and Occludin by Activating Angiopoietin-1 in Stroke Damage

This study examines the regulating effect of Sonic Hedgehog (Shh) on the permeability of the blood-brain barrier (BBB) in cerebral ischemia. By employing permanent middle cerebral artery occlusion (pMCAO) model, we find that Shh significantly decreases brain edema and preserves BBB permeability. Moreover, Shh increases zonula occludens-1 (ZO-1), occludin and angiopiotetin-1 (Ang-1) expression in the ischemic penumbra. Blockage of Shh with cyclopamine abolishes the effects of Shh on brain edema, BBB permeability and ZO-1, occludin, Ang-1 expression. Primary brain microvessel endothelial cells (BMECs) and astrocytes were pre-treated with Shh, cyclopamine, Ang-1-neutralizing antibody, and subjected to oxygen-glucose deprivation (OGD). Results show that the Ang-1 protein level in the culture medium of Shh-treated astrocytes is significantly higher. Shh also increased ZO-1, occludin and Ang-1 expression in BMECs, while cyclopamine and Ang-1-neutralizing antibody inhibited the effects of Shh on the ZO-1 and occludin expression, respectively. This study suggests that, under ischemic insults, Shh triggers Ang-1 production predominantly in astrocytes, and the secreted Ang-1 acts on BMECs, thereby upregulating ZO-1 and occludin to repair the tight junction and ameliorate the brain edema and BBB leakage.     

Mice lacking insulin or insulin-like growth factor 1 receptors in vascular endothelial cells maintain normal blood-brain barrier

The blood-brain barrier (BBB) is created by a combination of endothelial cells with tight junctions and astrocytes. One of the key tight junction proteins, zona occludens-1 (ZO-1), has been reported to be stimulated in its expression by insulin and IGF-1. To assess the role of insulin and IGF-1 in endothelial cells in the BBB we have utilized mice with a vascular endothelial cell-specific knockout of the insulin receptor (VENIRKO) and IGF-1 receptor (VENIFARKO). Both of these mice show a normal BBB based on no increase in leakage of Evans blue dye in the brain of these mice basally or after cold injury. Furthermore, the structural integrity of the BBB and blood-retinal barrier (BRB) was intact using the vascular markers lectin B-4 and ZO-1, and both proteins were properly co-localized in both brain and retinal vascular tissue of these mice. These observations indicate that neither insulin nor IGF-1 signaling in vascular endothelial cells is required for development and maintenance of BBB or BRB.     

Localization of occludin, ZO-1, and pan-cadherin in rabbit ciliary epithelium and iris vascular endothelium

Previous studies have used conventional electron microscopy and freeze fracture to identify the morphological equivalents of the blood-aqueous barrier in the mammalian eye. These equivalents are the tight junctions that form a part of the apicolateral junctional complex between adjacent non-pigmented ciliary epithelial cells and the tight junctions present between endothelial cells of the iris vasculature. Recent investigations have begun to unravel the molecular assembly of the tight junction and some variability has been found. Our goal in the present study was to probe the ciliary epithelium and iris vascular endothelium of the rabbit eye to determine if certain molecular constituents associated with tight junctions in other tissues are also present as parts of the blood-aqueous barrier. The selected constituents were occludin, ZO-1, and a representative, adherens junction-related cadherin. Immunohistochemical and immunoelectron microscopic methods were used. The results showed that occludin was distributed exclusively at known locations of tight junctions. ZO-1 was also expressed at these locations but its distribution extended beyond that of occludin, along the adjacent membranes. Pan-cadherin was expressed ubiquitously within the ciliary epithelium and negligibly in iris vascular endothelium. Our results demonstrate that occludin and ZO-1 are integral components of the blood-aqueous barrier of the normal rabbit eye.     

Restoration of tight junction structure and barrier function by down-regulation of the mitogen-activated protein kinase pathway in ras-transformed Madin-Darby canine kidney cells

In the Madin-Darby canine kidney epithelial cell line, the proteins occludin and ZO-1 are structural components of the tight junctions that seal the paracellular spaces between the cells and contribute to the epithelial barrier function. In Ras-transformed Madin-Darby canine kidney cells, occludin, claudin-1, and ZO-1 were absent from cell-cell contacts but were present in the cytoplasm, and the adherens junction protein E-cadherin was weakly expressed. After treatment of the Ras-transformed cells with the mitogen-activated protein kinase kinase (MEK1) inhibitor PD98059, which blocks the activation of mitogen-activated protein kinase (MAPK), occludin, claudin-1, and ZO-1 were recruited to the cell membrane, tight junctions were assembled, and E-cadherin protein expression was induced. Although it is generally believed that E-cadherin-mediated cell-cell adhesion is required for tight junction assembly, the recruitment of occludin to the cell-cell contact area and the restoration of epithelial cell morphology preceded the appearance of E-cadherin at cell-cell contacts. Both electron microscopy and a fourfold increase in the transepithelial electrical resistance indicated the formation of functional tight junctions after MEK1 inhibition. Moreover, inhibition of MAPK activity stabilized occludin and ZO-1 by differentially increasing their half-lives. We also found that during the process of tight junction assembly after MEK1 inhibition, tyrosine phosphorylation of occludin and ZO-1, but not claudin-1, increased significantly. Our study demonstrates that down-regulation of the MAPK signaling pathway causes the restoration of epithelial cell morphology and the assembly of tight junctions in Ras-transformed epithelial cells and that tyrosine phosphorylation of occludin and ZO-1 may play a role in some aspects of tight junction formation.     

Colocalization of tight junction proteins, occludin and ZO-1, and glucose transporter GLUT1 in cells of the blood-ocular barrier in the mouse eye

 The facilitative glucose transporter GLUT1 is abundant in cells of the blood-ocular barrier and serves as a glucose transport mechanism in the barrier. To see the relationship between the glucose transfer function and junctional proteins in the barrier, we examined the localization of GLUT1 and the tight junction proteins, occludin and ZO-1, in the mouse eye. Their localization in the retina, ciliary body, and iris was visualized by double-immunofluorescence microscopy and immunogold electron microscopy. Occludin and ZO-1 were colocalized at tight junctions of the cells of the barrier: retinal pigment epithelial cells, non-pigmented epithelial cells of the ciliary body, and endothelial cells of GLUT1-positive blood vessels. Occludin was restricted to these cells of the barrier. ZO-1 was found, in addition, in sites not functioning as a barrier: the outer limiting membrane in the retina, in the cell border between pigmented and non-pigmented epithelial cells in the ciliary body, and GLUT1-negative blood vessels. These observations show that localization of occludin is restricted to tight junctions of cells of the barrier, whereas ZO-1 is more widely distributed. Accepted: 7 September 1998     

Synergistic effects of neurons and astrocytes on the differentiation of brain capillary endothelial cells in culture

Brain capillary endothelial cells form a functional barrier between blood and brain, based on the existence of tight junctions that limit paracellular permeability. Occludin is one of the major transmembrane proteins of tight junctions and its peripheral localization gives indication of tight junction formation. We previously reported that RBE4.B cells (brain capillary endothelial cells), cultured on collagen IV, synthesize occludin and correctly localize it at the cell periphery only when cocultured with neurons. In the present study, we describe a three-cell type-culture system that allowed us to analyze the combined effects of neurons and astrocytes on differentiation of brain capillary endothelial cells in culture. In particular, we found that, in the presence of astrocytes, the neuron-induced synthesis and localization of occludin is precocious as compared to cells cocultured with neurons only.     

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 -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.This work is dedicated to the memory of Werner Risau (died 13.12.1998), who initiated this collaboration     

Tight Junctions of the Blood–Brain Barrier

1. The blood–brain barrier is essential for the maintainance and regulation of the neural microenvironment. The blood–brain barrier endothelial cells comprise an extremely low rate of transcytotic vesicles and a restrictive paracellular diffusion barrier. The latter is realized by the tight junctions between the endothelial cells of the brain microvasculature, which are subject of this review. Morphologically, blood–brain barrier-tight junctions are more similar to epithelial tight junctions than to endothelial tight junctions in peripheral blood vessels.2. Although blood–brain barrier-tight junctions share many characteristics with epithelial tight junctions, there are also essential differences. However, in contrast to tight junctions in epithelial systems, structural and functional characteristics of tight junctions in endothelial cells are highly sensitive to ambient factors.3. Many ubiquitous molecular constituents of tight junctions have been identified and characterized including claudins, occludin, ZO-1, ZO-2, ZO-3, cingulin, and 7H6. Signaling pathways involved in tight junction regulation comprise, among others, G-proteins, serine, threonine, and tyrosine kinases, extra- and intracellular calcium levels, cAMP levels, proteases, and TNF. Common to most of these pathways is the modulation of cytoskeletal elements which may define blood–brain barrier characteristics. Additionally, cross-talk between components of the tight junction– and the cadherin–catenin system suggests a close functional interdependence of the two cell–cell contact systems.4. Recent studies were able to elucidate crucial aspects of the molecular basis of tight junction regulation. An integration of new results into previous morphological work is the central intention of this review.     

Regulation of bovine brain microvascular endothelial tight junction assembly and barrier function by laminar shear stress

Blood-brain barrier (BBB) controls paracellular solute diffusion into the brain microenvironment and is maintained primarily by tight junctions between adjacent microvascular endothelial cells. Studies implicate blood flow-associated shear stress as a pathophysiological mediator of BBB function, although detailed biochemical data are scarce. We hypothesize that shear stress upregulates BBB function via direct modulation of expression and properties of pivotal tight-junction proteins occludin and zonula occludens-1 (ZO-1). Bovine brain microvascular endothelial cells (BBMvECs) were exposed to either steady or pulsatile shear stress (10 and 14 dyn/cm(2), respectively) for 24 h. Sheared BBMvECs were monitored for occludin-ZO-1 expression, association, and subcellular localization, and transendothelial permeability of BBMvECs to FITC-dextran and (14)[C]sucrose was assessed. Actin reorganization and BBMvEC realignment were observed following steady shear stress for 24 h. Substantial increases in occludin mRNA and protein expression (2.73 +/- 0.26- and 1.83 +/- 0.03-fold) and in occludin-ZO-1 association (2.12 +/- 0.15-fold) were also observed. Steady shear stress also induced clear relocalization of both proteins to the cell-cell border in parallel with reduced transendothelial permeability to FITC-dextran (but not sucrose). Following pulsatile shear stress, increased protein levels for both occludin and ZO-1 (2.15 +/- 0.02- and 1.67 +/- 0.21-fold) and increased occludin-ZO-1 association (2.91 +/- 0.14-fold) were observed in parallel with a reduction in transendothelial permeability to (14)[C]sucrose. Shear stress upregulates BBMvEC barrier function at the molecular level via modulation of expression, association, and localization of occludin and ZO-1. The pulsatile shear model appeared to give the most profound biochemical responses.     

Renal ischemia�Creperfusion injury causes intercalated cell-specific disruption of occludin in the collecting duct

Renal ischemic events open tight junctions and disrupt epithelial polarity. The purpose of this study was to examine the effects of ischemia-reperfusion (IR) injury on expression and distribution of the tight junction proteins, occludin and ZO-1, in the rat kidney. IR injury was induced by clamping both renal pedicles for 30 min and animals were killed at 6 h after the reperfusion. IR injury decreased blood bicarbonate level, but did not persistently alter pH, Na(+), K(+), or Cl(-). In control kidneys, occludin immunoreactivity was intense in the tight junctions in the thick ascending limb, distal convoluted tubule, and collecting duct, moderate in the thin limbs of the loop of Henle, and was not detected in the proximal tubule, glomerulus, and blood vessels. ZO-1 was expressed in the same sites in which occludin was expressed, and additionally was also expressed in the proximal tubule, glomerulus, and vascular endothelial cells. IR kidneys exhibited damaged renal tubular epithelial cells in both proximal tubule and collecting duct segments in the outer medulla. In the collecting duct, the response of intercalated cells and principal cells differed. Following IR injury, intercalated cells, but not principal cells, lost their normal epithelial polarity and were frequently extruded into the tubule lumen. Occludin, instead of being localized to tight junctions, was localized diffusely in the cytoplasm in intercalated cells of IR kidneys. Principal cells, in contrast, were not detectably affected and neither occludin nor ZO-1 expression were altered in response to IR injury. The normal localization of ZO-1 expression to tight junction sites in both the proximal tubule and collecting duct was altered in response to IR, and, instead, ZO-1 expression was present diffusely in the cytoplasm. IR injury did not alter detectably either occludin or ZO-1 localization to the tight junction of the thick ascending limb cells. The abundance of total occludin protein by immunoblot analysis was not changed with IR injury. These results demonstrate that renal IR injury causes tight junction disruptions in both the proximal tubule and the collecting duct, and that altered distribution of the tight junction protein, occludin, may play a critical role in the collecting duct dysfunction which IR induces.     

Culture of murine brain microvascular endothelial cells that maintain expression and cytoskeletal association of tight junction-associated proteins

A readily obtainable in vitro paradigm of the blood-brain barrier (BBB) would offer considerable benefits. Toward this end, in this study, we describe a novel method for purifying murine brain microvascular endothelial cells (BMEC) for culture. The method uses limited collagenase-dispase digestion of enriched brain microvessels, followed by immunoisolation of digested, microvascular fragments by magnetic beads coated with antibody to platelet-endothelial cell adhesion molecule-1. When plated onto collagen IV-coated surfaces, these fragments elaborated confluent monolayers of BMEC that expressed, as judged by immunocytochemistry, the adherens junction-associated proteins, VE-cadherin and beta-catenin, as well as the tight junction (TJ)-associated proteins, claudin-5, occludin, and zonula occludin-1 (ZO-1), in concentrated fashion along intercellular borders. In contrast, cultures of an immortalized and transformed line of murine brain capillary-derived endothelial cells, bEND.3, displayed diffuse cytoplasmic localization of occludin and ZO-1. This difference in occludin and ZO-1 staining between the two endothelial cell types was also reflected in the extent of association of these proteins with the detergent-resistant cytoskeletal framework (CSK). Although both occludin and ZO-1 largely partitioned with the CSK fraction in BMEC, they were found predominantly in the soluble fraction of bEND.3 cells, and claudin-5 was found associated equally with both fractions in BMEC and bEND.3 cells. Moreover, detergent-extracted cultures of the BMEC retained pronounced immunostaining of occludin and ZO-1, but not claudin-5, along intercellular borders. Because both occludin and ZO-1 are thought to be functionally coupled to the detergent-resistant CSK and high expression of TJs is considered a seminal characteristic of the BBB, these results impart that this method of purifying murine BMEC provides a suitable platform to investigate BBB properties in vitro.     

Biphasic effects of 17-beta-estradiol on expression of occludin and transendothelial resistance and paracellular permeability in human vascular endothelial cells

Tight junctions govern the paracellular permeability of endothelial and epithelial cells. Aberrations of tight junction function are an early and key event during the vascular spread of cancer and inflammation. This study sought to determine the role of estrogen in the regulation of tight junctions and expression of molecules making tight junctions in endothelial cells. Human endothelial cell, HECV, which express ER-beta but not ER-alpha was used. 17-beta-estradiol induced a concentration- and time-dependent biphasic effect on tight junction. At 10(-9) and 10(-6) M, it decreased the level of occludin and increased in paracellular permeability of HECV cells, but at 10(-12) M it decreased in paracellular permeability and increased the level of occludin. The transendothelial electrical resistance (TER), however, was reduced by 17-beta-estradiol at lower concentrations (as low as 10(-12) M). Furthermore, the time-dependent biphasic effect was observed over a period of 4 days, with the first reduction of TER seen within 15 min and the second drop occurring 48 h after 17-beta-estradiol treatment. It was further revealed that protein and mRNA levels of occludin, but not claudin-1 and -5, and ZO-1, were reduced by 17-beta-estradiol, in line with changes of TER. This study shows that 17-beta-estradiol can induce concentration- and time-related biphasic effects on tight junction functions expression of occludin in endothelial cells and that this perturbation of tight junction functions may have implications in the etiology of mastalgia and the vascular spread of breast cancer.     

Bile duct ligation in the rat causes upregulation of ZO-2 and decreased colocalization of claudins with ZO-1 and occludin

As the only barrier between blood and bile compartments hepatocellular tight junctions play a crucial role in cholestasis-induced increase of biliary permeability. The molecular basis of this reversible defect is not known. We, therefore, examined expression, phosphorylation, distribution and colocalization of the junctional proteins occludin, claudin-1-3, ZO-1 and ZO-2 in rats after bile duct ligation and release of ligation. In control rats, claudin-1 and ZO-2 displayed a lobular gradient with highest expression levels in periportal cells, whereas claudin-2 showed a reciprocal distribution. Other proteins were evenly expressed in the liver lobule. Ligation resulted in upregulation of ZO-2 (2.7-fold), ZO-1 (1.4-fold) and occludin (1.2-fold) but not of claudins. Only ZO-2 showed increased phosphorylation. Distribution patterns were unchanged except for a strong accumulation of ZO-2 in perivenous hepatocytes. Colocalization analysis demonstrated that perivenous ZO-2 was the only protein examined revealing strongly increased overlap with occludin and ZO-1, whereas claudins and other proteins displayed a decrease. All changes were partially reversed by release of ligation. We conclude that differential expression of claudin-1-2 and ZO-2 has functional implications for bile formation. The moderately increased ZO-1 and occludin levels account for the known elongation of tight junction strands. The highly increased expression and changed distribution of ZO-2 suggests that ZO-1 is partly substituted by ZO-2, an alteration possibly causing impaired barrier function.     

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.