Tight junctions contain oligomeric protein assembly critical for maintaining blood-brain barrier integrity in vivo

Tight junctions (TJs) are major components of the blood–brain barrier (BBB) that physically obstruct the interendothelial space and restrict paracellular diffusion of blood-borne substances from the peripheral circulation to the CNS. TJs are dynamic structures whose intricate arrangement of oligomeric transmembrane and accessory proteins rapidly alters in response to external stressors to produce changes in BBB permeability. In this study, we investigate the constitutive trafficking of the TJ transmembrane proteins occludin and claudin-5 that are essential for forming the TJ seal between microvascular endothelial cells that inhibits paracellular diffusion. Using a novel, detergent-free OptiPrep density-gradient method to fractionate rat cerebral microvessels, we identify a plasma membrane lipid raft domain that contains oligomeric occludin and claudin-5. Our data suggest that oligomerization of occludin involves disulfide bond formation within transmembrane regions, and that assembly of the TJ oligomeric protein complex is facilitated by an oligomeric caveolin scaffold. This is the first time that distribution of oligomeric TJ transmembrane proteins within plasma membrane lipid rafts at the BBB has been examined in vivo. The findings reported in this study are critical to understand the mechanism of assembly of the TJ multiprotein complex that is essential for maintaining BBB integrity.     

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.     

Tempol modulates changes in xenobiotic permeability and occludin oligomeric assemblies at the blood-brain barrier during inflammatory pain

Our laboratory has shown that λ-carrageenan-induced peripheral inflammatory pain (CIP) can alter tight junction (TJ) protein expression and/or assembly leading to changes in blood-brain barrier xenobiotic permeability. However, the role of reactive oxygen species (ROS) and subsequent oxidative stress during CIP is unknown. ROS (i.e., superoxide) are known to cause cellular damage in response to pain/inflammation. Therefore, we examined oxidative stress-associated effects at the blood-brain barrier (BBB) in CIP rats. During CIP, increased staining of nitrosylated proteins was detected in hind paw tissue and enhanced presence of protein adducts containing 3-nitrotyrosine occurred at two molecular weights (i.e., 85 and 44 kDa) in brain microvessels. Tempol, a pharmacological ROS scavenger, attenuated formation of 3-nitrotyrosine-containing proteins in both the hind paw and in brain microvessels when administered 10 min before footpad injection of λ-carrageenan. Similarly, CIP increased 4-hydroxynoneal staining in brain microvessels and this effect was reduced by tempol. Brain permeability to [(14)C]sucrose and [(3)H]codeine was increased, and oligomeric assemblies of occludin, a critical TJ protein, were altered after 3 h CIP. Tempol attenuated both [(14)C]sucrose and [(3)H]codeine brain uptake as well as protected occludin oligomers from disruption in CIP animals, suggesting that ROS production/oxidative stress is involved in modulating BBB functional integrity during pain/inflammation. Interestingly, tempol administration reduced codeine analgesia in CIP animals, indicating that oxidative stress during pain/inflammation may affect opioid delivery to the brain and subsequent efficacy. Taken together, our data show for the first time that ROS pharmacological scavenging is a viable approach for maintaining BBB integrity and controlling central nervous system drug delivery during acute inflammatory pain.     

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 ~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.     

Comparative changes in the blood-brain barrier and cerebral infarction of SHR and WKY rats

Hypertension is involved in the exacerbation of stroke. It is unclear how blood-brain barrier (BBB) tight-junction (TJ) and ion transporter proteins critical for maintaining brain homeostasis contribute to cerebral infarction during hypertension development. In the present study, we investigated cerebral infarct volume following permanent 4-h middle cerebral artery occlusion (MCAO) and characterized the expression of BBB TJ and ion transporter proteins in brain microvessels of spontaneously hypertensive rats (SHR) compared with age-matched Wistar-Kyoto (WKY) rats at 5 wk (prehypertension), 10 wk (early-stage hypertension), and 15 wk (later-stage hypertension) of age. Hypertensive SHR show increased infarct volume following MCAO compared with WKY control rats. BBB TJ and ion transporter proteins, known to contribute to edema and fluid volume changes in the brain, show differential protein expression patterns during hypertension development. Western blot analysis of TJ protein zonula occludens-2 (ZO-2) showed decreased expression, while ion transporter, Na(+)/H(+) exchanger 1 (NHE-1), was markedly increased in hypertensive SHR. Expression of TJ proteins ZO-1, occludin, actin, claudin-5, and Na(+)-K(+)-2Cl(-) cotransporter remain unaffected in SHR compared with control. Selective inhibition of NHE-1 using dimethylamiloride significantly attenuated ischemia-induced infarct volume in hypertensive SHR following MCAO, suggesting a novel role for NHE-1 in the brain in the regulation of ischemia-induced infarct volume in SHR.     

Cerebral microvascular changes in permeability and tight junctions induced by hypoxia-reoxygenation

Cerebral microvessel endothelial cells that form the blood-brain barrier (BBB) have tight junctions (TJ) that are critical for maintaining brain homeostasis and low permeability. Both integral (claudin-1 and occludin) and membrane-associated zonula occluden-1 and -2 (ZO-1 and ZO-2) proteins combine to form these TJ complexes that are anchored to the cytoskeletal architecture (actin). Disruptions of the BBB have been attributed to hypoxic conditions that occur with ischemic stroke, pathologies of decreased perfusion, and high-altitude exposure. The effects of hypoxia and posthypoxic reoxygenation in cerebral microvasculature and corresponding cellular mechanisms involved in disrupting the BBB remain unclear. This study examined hypoxia and posthypoxic reoxygenation effects on paracellular permeability and changes in actin and TJ proteins using primary bovine brain microvessel endothelial cells (BBMEC). Hypoxia induced a 2.6-fold increase in [(14)C]sucrose, a marker of paracellular permeability. This effect was significantly reduced (~58%) with posthypoxic reoxygenation. After hypoxia and posthypoxic reoxygenation, actin expression was increased (1.4- and 2.3-fold, respectively). Whereas little change was observed in TJ protein expression immediately after hypoxia, a twofold increase in expression was seen with posthypoxic reoxygenation. Furthermore, immunofluorescence studies showed alterations in occludin, ZO-1, and ZO-2 protein localization during hypoxia and posthypoxic reoxygenation that correlate with the observed changes in BBMEC permeability. The results of this study show hypoxia-induced changes in paracellular permeability may be due to perturbation of TJ complexes and that posthypoxic reoxygenation reverses these effects.     

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.     

Immunolocalization of tight junction proteins in the adult and developing human brain

The formation of endothelial tight junctions (TJs) is crucial in blood-brain barrier (BBB) differentiation, and the expression and targeting of TJ-associated proteins mark the beginning of BBB functions. Using confocal microscopy, this study analyzed endothelial TJs in adult human cerebral cortex and the fetal telencephalon and leptomeninges in order to compare the localization of two TJ-associated transmembrane proteins, occludin and claudin-5. In the arterioles and microvessels of adult brain, occludin and claudin-5 form continuous bands of endothelial immunoreactivity. During fetal development, occludin and claudin-5 immunoreactivity is first detected as a diffuse labeling of endothelial cytoplasm. Later, at 14 weeks, the immunosignal for both proteins shifts from the cytoplasm to the interface of adjacent endothelial cells, forming a linear, widely discontinuous pattern of immunoreactivity that achieves an adult-like appearance within a few weeks. These results demonstrate that occludin and claudin-5 expression is an early event in human brain development, followed shortly by assembly of both proteins at the junctional areas. This incremental process suggests more rapid establishment of the human BBB, consistent with its specific function of creating a suitable environment for neuron differentiation and neurite outgrowth during neocortical histogenesis.Electronic Supplementary Material Supplementary material is available in the online version of this article at http://dx.doi.org/10.1007/s00418-004-0665-1Daniela Virgintino and Mariella Errede contributed equally to this work     

Tight junction proteins in gallbladder epithelium: different expression in acute acalculous and calculous cholecystitis.

There is a paucity of information of tight junction (TJ) proteins in gallbladder epithelium, and disturbances in the structure of these proteins may play a role in the pathogenesis of acute acalculous cholecystitis (AAC) and acute calculous cholecystitis (ACC). Using immunohistochemistry, we investigated the expression of TJ proteins claudin-1, -2, -3, and -4, occludin, zonula occludens (ZO-1), and E-cadherin in 9 normal gallbladders, 30 gallbladders with AAC, and 21 gallbladders with ACC. The number of positive epithelial and endothelial cells and the intensity of the immunoreaction were determined. Membrane-bound and cytoplasmic immunoreactivities were separately assessed. We found that TJ proteins were uniformly expressed in normal gallbladder epithelium, with the exception of claudin-2, which was present in less than half of the cells. In AAC, expression of cytoplasmic occludin and claudin-1 were decreased, as compared with normal gallbladder. In ACC, expression of claudin-2 was increased, and expression of claudin-1, -3, and -4, occludin, and ZO-1 were decreased, as compared with normal gallbladder or AAC. We conclude that there are significant differences in expression of TJ proteins in AAC and ACC, supporting the idea that AAC represents a manifestation of systemic inflammatory disease, whereas ACC is a local inflammatory and often infectious disease.     

Effect of n-3 polyunsaturated fatty acids on membrane microdomain localization of tight junction proteins in experimental colitis

Ulcerative colitis (UC) is a gastrointestinal disorder characterized by an inflammatory process associated with mucosal damage. Many studies have shown that n-3 polyunsaturated fatty acids (PUFAs) possess anti-inflammatory effects in inflammatory bowel disease. The aim of this study was to investigate whether n-3 PUFAs could alleviate intestinal damage in experimental UC. In the present study, we found that in 2,4,6-trinitrobenzenesulfonic acid-induced colitic rats, the damage to the intestinal mucosa was accompanied by a disrupted tight junction (TJ) structure. In accordance with these changes, the distribution and expression of TJ proteins, including occludin, claudin-1, claudin-3, claudin-5, claudin-8 and ZO-1, in membrane microdomains was altered. The distribution of flotillin-1, a lipid raft marker protein, was also changed. Moreover, we found for the first time that n-3 PUFAs prevented redistribution of TJ proteins from Triton X-100-insoluble raft-like membrane microdomains to Triton X-100-soluble fractions. The expression of ZO-1, claudin-1, claudin-5 and claudin-8 was significantly elevated by n-3 PUFAs. n-3 PUFAs also attenuated the disruption of TJ structure and improved the histological score. Our results demonstrate that the expression and distribution of TJ proteins in TJ membrane microdomains might be affected in UC, and that such altered expression of TJ proteins in membrane microdomains in experimental UC is affected by n-3 PUFAs. These findings may have therapeutic potential in intestinal inflammation.     

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     

Immunogold localization of tight junctional proteins in normal and osmotically-affected rat blood-brain barrier

The distribution of molecular components of interendothelial tight junctions (TJs) was studied in rat blood-brain barrier (BBB) microvessels, using immunogold cytochemistry applied to electron microscopy. Samples of rat brains, both normal (unaffected) and osmotically-affected (1, 5, and 30 min after intracarotid infusion of 1.8 M L(+)arabinose), were processed for immunocytochemical localization of TJ-specific integral membrane (occludin, JAM-1, claudin-5) and peripheral (ZO-1) protein molecules. In unaffected interendothelial junctions of control rats the immunosignals (represented by gold particles) for occludin and ZO-1 were of highest, whereas for claudin-5 and JAM-1 were of lower density. At 1 min after infusion, no discernible changes in distribution of junction-associated molecules were noted. At 5 min, however, changes were most conspicuous, and they consisted of segmental attenuation of the endothelial lining and dilatation (opening) of some junctional clefts accompanied by the diminution of the density of immunosignals for TJ-specific transmembrane and peripheral proteins. It was paralleled by disorganization of the spatial relation of these molecules to the junctional complexes. After 30 min, many interendothelial junctions appeared to be still open, whereas other junctions were partially or totally closed. In the opened interendothelial junctions the expression of TJ-associated molecules was weaker than in closed junctions. Our observations indicate that the localization and expression of TJ-specific proteins, especially occludin, and in lower degree claudin-5 and JAM-1, together with the peripheral ZO-1 molecules, are affected by osmotic shock. Presumably, some of these proteins (e.g., occludin, claudin-5 and ZO-1) could be considered sensitive indicators of normal and also of disturbed functional state of the BBB.     

Kidney claudin-19: localization in distal tubules and collecting ducts and dysregulation in polycystic renal disease

Tight junction (TJ) constitutes the barrier by controlling the passage of ions and molecules via paracellular pathway and the movement of proteins and lipids between apical and basolateral domains of the plasma membrane. Claudins, occludin, and junctional adhesion molecules are the major three transmembrane proteins at TJ. This study focuses a newly identified mammalian TJ gene, claudin-19, in kidneys. Mouse claudin-19 composes of 224 amino acids and shares 98.2% and 95% amino acid homology with rat and human, respectively; the most evolutionary-related claudins are claudin-1 and -7, which share approximately 75% DNA sequence homology with claudin-19. Claudin-19 is abundantly expressed in the mouse and rat kidneys among the organs examined by Northern blots, and to a much less extent, also found in brain by RT-PCR. Claudin-19 and zonula occludens-1 (ZO-1) are localized at junctional regions of Madin-Darby canine kidney (MDCK) cells by immunofluorescent microscopy. In addition, ZO-1 is found in the claudin-19-associated protein complexes in MDCK cells by co-immunoprecipitation. Using aquaporin-1 and aquaporin-2 antibodies as markers for different renal segment, strong expression of claudin-19 was observed in distal tubules of the cortex as well as in the collecting ducts of the medulla. To less extent, claudin-19 is also present in the proximal tubules (cortex) and in the loop of Henle (medulla). Furthermore, intense claudin-19 immunoreactivity is found co-localized with the ZO-1 in kidneys from postnatal day 15, day 45, and adult rats and mice. Similar localizations of claudin-19 and ZO-1 are also observed in human kidneys. Since these renal segments are mainly for controlling the paracellular cation transport, it is suggested that claudin-19 may participate in these processes. In human polycystic kidneys, decreased expression and dyslocalization of claudin-19 are noticed, suggesting a possible correlation between claudin-19 and renal disorders. Taken together, claudin-19 is a claudin isoform that is highly and specifically expressed in renal tubules with a putative role in TJ homeostasis in renal physiology.     

Caveolae-mediated Internalization of Occludin and Claudin-5 during CCL2-induced Tight Junction Remodeling in Brain Endothelial Cells

Disturbance of the tight junction (TJ) complexes between brain endothelial cells leads to increased paracellular permeability, allowing leukocyte entry into inflamed brain tissue and also contributing to edema formation. The current study dissects the mechanisms by which a chemokine, CCL2, induces TJ disassembly. It investigates the potential role of selective internalization of TJ transmembrane proteins (occludin and claudin-5) in increased permeability of the brain endothelial barrier in vitro. To map the internalization and intracellular fate of occludin and claudin-5, green fluorescent protein fusion proteins of these TJ proteins were generated and imaged by fluorescent microscopy with simultaneous measurement of transendothelial electrical resistance. During CCL2-induced reductions in transendothelial electrical resistance, claudin-5 and occludin became internalized via caveolae and further processed to early (EEA1+) and recycling (Rab4+) endosomes but not to late endosomes. Western blot analysis of fractions collected from a sucrose gradient showed the presence of claudin-5 and occludin in the same fractions that contained caveolin-1. For the first time, these results suggest an underlying molecular mechanism by which the pro-inflammatory chemokine CCL2 mediates brain endothelial barrier disruption during CNS inflammation.The blood-brain barrier is situated at the cerebral endothelial cells and their linking tight junctions. Increased brain endothelial barrier permeability is associated with remodeling of inter-endothelial tight junction (TJ)² complex and gap formation between brain endothelial cells (paracellular pathway) and/or intensive pinocytotic vesicular transport between the apical and basal side of brain endothelial cells (transcellular pathway) (1, 2). The transcellular pathway can be either passive or active and is characterized by low conductance and high selectivity. In contrast, the paracellular pathway is exclusively passive, being driven by electrochemical and osmotic gradients, and has a higher conductance and lower selectivity (3).Brain endothelial barrier paracellular permeability is maintained by an equilibrium between contractile forces generated at the endothelial cytoskeleton and adhesive forces produced at endothelial cell-cell junctions and cell-matrix contacts (1–3). A dynamic interaction among these structural elements controls opening and closing of the paracellular pathway and serves as a fundamental mechanism regulating blood-brain exchange. How this process occurs is under intense investigation. Two possible mechanisms may potentially increase paracellular permeability: phosphorylation of TJ proteins and/or endocytosis of transmembrane TJ proteins.Changes in TJ protein phosphorylation seem to be required to initiate increased brain endothelial permeability and a redistribution of most TJ proteins away from the cell border (4–8). Endocytosis may also be involved in remodeling TJ complexes between endothelial cells. Several types of endocytosis may be involved in TJ protein uptake, including clathrin- and caveolae-mediated endocytosis and macropinocytosis (for reviews, see Refs. 8 and 9–12). After first forming cell membrane-derived endocytotic vesicles, these vesicles fuse with early endosomes whose contents are further sorted for transport to lysosomes for degradation or recycling back to the plasma membrane for reuse (11).Although there is a lack of definitive knowledge regarding endocytotic internalization of brain endothelial cell TJ proteins, several studies on epithelial cells have indicated that occludin may be internalized via caveolae-mediated endocytosis whereas ZO-1, claudin-1, and junctional adhesion molecules-A may undergo macropinocytosis in response to stimuli such as TNF-α and INF-γ (13, 14). In contrast, there is evidence that Ca²⁺ may induce internalization of claudin-1 and occludin via clathrin-coated vesicles (8, 14–16). All of these studies pinpoint endocytosis as an underlying process in TJ complex remodeling and redistribution, and thus regulation of paracellular permeability in epithelial cells.The present study examines whether internalization of transmembrane TJ proteins could be one process by which adhesion between brain endothelial cells is changed during increased paracellular permeability. Our results show that a pro-inflammatory mediator, the chemokine CCL2, induces disassembly of the TJ complex by triggering caveolae-dependent internalization of transmembrane TJ proteins (occludin and claudin-5). Once internalized, occludin and claudin-5 are further processed to recycling endosomes awaiting return to the plasma membrane.     

Organization and formation of the tight junction system in human epidermis and cultured keratinocytes

Occludin and several proteins of the claudin family have been identiried in simple epithelia and in endothelia as major and structure-determining transmembrane proteins clustered in the barrier-forming tight junctions (TJ), where they are associated with a variety of TJ plaque proteins, including protein ZO-1. To examine whether TJ also occur in the squamous stratified epithelium of the interfollicular human epidermis we have applied several microscopic and biochemical techniques. Using RT-PCR techniques, we have identiried mRNAs encoding protein ZO-1, occludin and claudins 1, 4, 7, 8, 11, 12, and 17 in both tissues, skin and cultured keratinocytes, whereas claudins i and 10 have only been detected in skin tissue. By immunocytochemistry we have localized claudin-1, occludin and protein ZO-1 in distinct plasma membrane structures representing cell-cell attachment zones. While claudin-1 occurs in plasma membranes of all living cell layers, protein ZO-1 is concentrated in or even restricted to the uppermost layers, and occludin is often detected only in the stratum granulosum. Using electron microscopy, typical TJ structures ("kissing points") as well as some other apparently related junctional structures have been detected in the stratum granulosum, interspersed between desmosomes. Modes and patterns of TJ formation have also been studied in experimental model systems, e.g., during wound healing and stratification as well as in keratinocyte cultures during Ca2+-induced stratification. We conclude that the epidermis contains in the stratum granulosum a continuous zonula occludens-equivalent structure with typical TJ morphology and molecular composition, characterized by colocalization of occludin, claudins and TJ plaque proteins. In addition, cell-cell contact structures and certain TJ proteins can also be detected in other epidermal cell layers in specific cell contacts. The pattern of formation and possible functions of epidermal TJ and related structures are discussed.     

Glutamine regulates Caco-2 cell tight junction proteins

Intestinal epithelial tight junction (TJ) barrier dysfunction may lead to inflammation and mucosal injury. Glutamine (GLN) plays a role in maintenance of intestinal barrier function in various animal models and critically ill humans. Recent evidence from intestinal cell monolayers indicates that GLN maintains transepithelial resistance and decreases permeability. The mechanisms of these effects remain undefined. We hypothesized that GLN affects proteins involved in the intercellular junctional complex. GLN availability was controlled in Caco-2 monolayers by addition to the medium and treatment with methionine sulfoximine (MSO) to inhibit glutamine synthetase (GS). Expression of TJ proteins, claudin-1, occludin, and zonula occluden (ZO)-1 was measured by immunoblotting. Localization of TJ proteins was evaluated by immunofluorescence light microscopy. Structure of TJ was determined by transmission electron microscopy (TEM). Deprivation of GLN decreased claudin-1, occludin, and ZO-1 protein expression and caused a disappearance of perijunctional claudin-1 and a reduction of occludin but had no effect on ZO-1. TEM revealed that MSO-treated cells in the absence of GLN formed irregular junctional complexes between the apical lateral margins of adjoining cells. These findings indicate that TJ protein expression and cellular localization in Caco-2 cell monolayers rely on GLN. This mechanism may similarly relate to GLN-mediated modulation of intestinal barrier function in stressed animals and humans.     

Actin depolymerization disrupts tight junctions via caveolae-mediated endocytosis

The tight junction (TJ) determines epithelial barrier function. Actin depolymerization disrupts TJ structure and barrier function, but the mechanisms of this effect remain poorly understood. The goal of this study was to define these mechanisms. Madin-Darby canine kidney (MDCK) cells expressing enhanced green fluorescent protein-, enhanced yellow fluorescent protein-, or monomeric red fluorescent protein 1-fusion proteins of beta-actin, occludin, claudin-1, ZO-1, clathrin light chain A1, and caveolin-1 were imaged by time-lapse multidimensional fluorescence microscopy with simultaneous measurement of transepithelial electrical resistance (TER). Actin depolymerization was induced with latrunculin A (LatA). Within minutes of LatA addition TER began to fall. This coincided with occludin redistribution and internalization. In contrast, ZO-1 and claudin-1 redistribution occurred well after maximal TER loss. Occludin internalization and TER loss, but not actin depolymerization, were blocked at 14 degrees C, suggesting that membrane traffic is required for both events. Inhibition of membrane traffic with 0.4 M sucrose also blocked occludin internalization and TER loss. Internalized occludin colocalized with caveolin-1 and dynamin II, but not with clathrin, and internalization was blocked by dominant negative dynamin II (K44A), but not by Eps15Delta95-295 expression. Inhibition of caveolae-mediated endocytosis by cholesterol extraction prevented both LatA-induced TER loss and occludin internalization. Thus, LatA-induced actin depolymerization causes TJ structural and functional disruption by mechanisms that include caveolae-mediated endocytosis of TJ components.     

Oxidative stress induces claudin-2 nitration in experimental type 1 diabetic nephropathy

Renal complications in diabetes are severe and may lead to renal insufficiency. Early alterations in tight junction (TJ) proteins in diabetic nephropathy (DN) have not been explored and the role of oxidative stress in their disassembly has been poorly characterized. We investigated the expression and distribution of TJ proteins: claudin-5 in glomeruli (GL), occludin and claudin-2 in proximal tubules (PTs), and ZO-1 and claudin-1, -4, and -8 in distal tubules (DTs) of rats 21 days after streptozotocin injection. Redox status along the nephron segments was evaluated. Diabetes increased kidney injury molecule-1 expression. Expression of sodium glucose cotransporters (SGLT1 and SGLT2) and facilitative glucose transporter (GLUT2) was induced. Increased oxidative stress was present in GL and PTs and to a lesser extent in DTs (measured by superoxide production and PKCβ2 expression), owing to NADPH oxidase activation and uncoupling of the endothelial nitric oxide synthase-dependent pathway. Claudin-5, occludin, and claudin-2 expression was decreased, whereas claudin-4 and -8 expression increased. ZO-1 was redistributed from membrane to cytosol. Increased nitration of tyrosine residues in claudin-2 was found, which might contribute to decrement of this protein in proximal tubule. In contrast, occludin was not nitrated. We suggest that loss of claudin-2 is associated with increased natriuresis and that loss of glomerular claudin-5 might explain early presence of proteinuria. These findings suggest that oxidative stress is related to alterations in TJ proteins in the kidney that are relevant to the pathogenesis and progression of DN and for altered sodium regulation in diabetes.     

Dynamics of tight and adherens junctions under EGTA treatment

The dynamics of tight junctions (TJs) and adherens junctions (AJs) under EGTA treatment were investigated in Madin Darby canine kidney (MDCK) cells. Detailed information about the behavior of TJ and AJ proteins during the opening and resealing of TJs and AJs is still scarce. By means of the "calcium chelation" method, the distribution and colocalization of junctional proteins were studied with confocal laser scanning microscopy using a deconvolution algorithm for high-resolution images. Colocalization was analyzed for pairs of the following proteins: ZO-1, occludin, claudin-1, E-cadherin and F-actin. Significant differences were found for the analyzed pairs in control cells compared to EGTA-treated cells with respect to the position of the colocalization maxima within the cell monolayers as well as with respect to the amount of colocalized voxels. Under EGTA treatment, colocalization for ZO-1/occludin, ZO-1/claudin-1, claudin-1/occludin, E-cadherin/occludin and E-cadherin/claudin-1 dropped below 35% of the control value. Only for the ZO-1/E-cadherin pair, the amount of colocalized voxels increased and a shift to a more basal position was observed. During the opening of TJs and AJs, ZO-1 colocalized with E-cadherin in the lateral membrane region, whereas in controls, ZO-1 colocalized with occludin and claudin-1 in the junctional complex. The combination of deconvolution with colocalization analysis of confocal data sets offers a powerful tool to investigate the spatial relationship of TJ and AJ proteins during assembly and disassembly of cell-cell contacts.     

Blood-brain barrier tight junctions are altered during a 72-h exposure to lambda-carrageenan-induced inflammatory pain

In this study, we examined the effect of lambda-carrageenan-induced inflammatory pain on the functional and structural properties of the rat blood-brain barrier (BBB) over a 72-h time period. Systemic inflammation was induced by an intraplantar injection of 3% lambda-carrageenan into the right hind paw of female Sprague-Dawley rats. In situ brain perfusion and Western blot analyses were performed at 1, 3, 6, 12, 24, 48, and 72 h. In situ brain perfusion showed lambda-carrageenan significantly increased brain uptake of [(14)C]sucrose at 1, 3, 6, and 48 h (139 +/- 9%, 166 +/- 19%, 138 +/- 13%, and 146 +/- 7% compared with control, respectively). Capillary depletion analysis insured the increased brain uptake was due to increased BBB permeability and not vascular trapping. Western blot analyses for zonula occludens-1 (ZO-1) and occludin were performed on isolated cerebral microvessels. ZO-1 expression was significantly increased at 1, 3, and 6 h and returned to control expression levels by 12 h. Total occludin expression was significantly reduced at 1, 3, 6, 12, and 48 h. This investigation demonstrated that lambda-carrageenan-induced inflammatory pain elicits a biphasic increase in BBB permeability with the first phase occurring from 1-6 h and the second phase occuring at 48 h. Furthermore, changes in BBB function are correlated with altered tight junctional protein expression of occludin and ZO-1. Changes in the structure of tight junctions may have important clinical ramifications concerning central nervous system homeostasis and therapeutic drug delivery.