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.     

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.     

Expression and function of tight junctions in the crypt epithelium of human palatine tonsils.

The human palatine tonsils have surface and crypt stratified epithelium and may be initiated via the epithelium to mount immune responses to various presenting antigens. Here we investigated the expression and function of tight junctions in the epithelium of human palatine tonsils from patients with tonsillar hypertrophy or recurrent tonsillitis. Occludin, ZO-1, JAM-1, and claudin-1, -3, -4, -7, -8, and -14 mRNAs were detected in tonsillar hypertrophy. Occludin and claudin-14 were expressed in the uppermost layer of the tonsil surface epithelium, whereas ZO-1, JAM-1, and claudin-1, -4, and -7 were found throughout the epithelium. In the crypt epithelium, claudin-4 was preferentially expressed in the upper layers. In freeze-fracture replicas, short fragments of continuous tight junction strands were observed but never formed networks. In the crypt epithelium of recurrent tonsillitis, the tracer was leaked from the surface regions where occludin and claudin-4 disappeared. Occludin, ZO-1, JAM-1, and claudin-1, -3, -4, and -14, but not claudin-7, mRNAs were decreased in recurrent tonsillitis compared with those of tonsillar hypertrophy. These studies suggest unique expression of tight junctions in human palatine tonsillar epithelium, and the crypt epithelium may possess an epithelial barrier different from that of the surface epithelium.     

Exogenous expression of the amino-terminal half of the tight junction protein ZO-3 perturbs junctional complex assembly

The functional characteristics of the tight junction protein ZO-3 were explored through exogenous expression of mutant protein constructs in MDCK cells. Expression of the amino-terminal, PSD95/dlg/ZO-1 domain-containing half of the molecule (NZO-3) delayed the assembly of both tight and adherens junctions induced by calcium switch treatment or brief exposure to the actin-disrupting drug cytochalasin D. Junction formation was monitored by transepithelial resistance measurements and localization of junction-specific proteins by immunofluorescence. The tight junction components ZO-1, ZO-2, endogenous ZO-3, and occludin were mislocalized during the early stages of tight junction assembly. Similarly, the adherens junction proteins E-cadherin and beta-catenin were also delayed in their recruitment to the cell membrane, and NZO-3 expression had striking effects on actin cytoskeleton dynamics. NZO-3 expression did not alter expression levels of ZO-1, ZO-2, endogenous ZO-3, occludin, or E-cadherin; however, the amount of Triton X-100-soluble, signaling-active beta-catenin was increased in NZO-3-expressing cells during junction assembly. In vitro binding experiments showed that ZO-1 and actin preferentially bind to NZO-3, whereas both NZO-3 and the carboxy-terminal half of the molecule (CZO-3) contain binding sites for occludin and cingulin. We hypothesize that NZO-3 exerts its dominant-negative effects via a mechanism involving the actin cytoskeleton, ZO-1, and/or beta-catenin.     

Lipopolysaccharide disrupts tight junctions in cholangiocyte monolayers by a c-Src-, TLR4-, and LBP-dependent mechanism

Bile duct epithelium forms a barrier to the backflow of bile into the liver parenchyma. However, the structure and regulation of the tight junctions in bile duct epithelium is not well understood. In the present study, we evaluated the effect of lipopolysaccharide on tight junction integrity and barrier function in normal rat cholangiocyte monolayers. Lipopolysaccharide disrupts barrier function and increases paracellular permeability in a time- and dose-dependent manner. Lipopolysaccharide induced a redistribution of tight junction proteins, occludin, claudin-1, claudin-4, and zonula occludens (ZO)-1 from the intercellular junctions and reduced the level of ZO-1. Tyrosine kinase inhibitors (genistein and PP2) prevented lipopolysaccharide-induced increase in permeability and subcellular redistribution of ZO-1. Reduced expression of c-Src, TLR4, or LBP by specific small interfering RNA attenuated lipopolysaccharide-induced permeability and redistribution of ZO-1. ML-7, a myosin light chain kinase inhibitor, attenuated LPS-induced permeability. Lipopolysaccharide treatment rapidly increased the phosphorylation of occludin and ZO-1 on tyrosine residues, which was prevented by genistein and PP2. Occludin and ZO-1 were found to be highly phosphorylated on threonine residues in intact cell monolayers. Threonine-phosphorylation of occludin was rapidly reduced by lipopolysaccharide administration. Lipopolysaccharide-induced dephosphorylation of occludin on Thr residues was prevented by genistein and PP2. In conclusion, lipopolysaccharide disrupts the tight junction of a bile duct epithelial monolayer by a c-Src-, TLR4-, LBP-, and myosin light chain kinase-dependent mechanism.     

Rapid disruption of intestinal barrier function by gliadin involves altered expression of apical junctional proteins

Coeliac disease is a chronic enteropathy caused by the ingestion of wheat gliadin and other cereal prolamines derived from rye and barley. In the present work, we investigated the mechanisms underlying altered barrier function properties exerted by gliadin-derived peptides in human Caco-2 intestinal epithelial cells. We demonstrate that gliadin alters barrier function almost immediately by decreasing transepithelial resistance and increasing permeability to small molecules (4 kDa). Gliadin caused a reorganisation of actin filaments and altered expression of the tight junction proteins occludin, claudin-3 and claudin-4, the TJ-associated protein ZO-1 and the adherens junction protein E-cadherin.     

Enhancement of cell-cell contact by claudin-4 in renal epithelial Madin-Darby canine kidney cells

Claudin-4 regulates ion permeability via a paracellular pathway in renal epithelial cells, but its other physiological functions have not been examined. We found that hyperosmotic stress increases claudin-4 expression in Madin-Darby canine kidney cells. Here, we examined whether claudin-4 affects cell motility, cell association, and the intracellular distribution of endogenous junctional proteins. Doxycycline-inducible expression of claudin-4 did not change endogenous levels of claudin-1, claudin-2, claudin-3, occludin, E-cadherin, and ZO-1. Claudin-4 overexpression increased cell association and decreased cell migration without affecting cell proliferation. Doxycycline did not change cell junctional protein levels, cell association or cell migration in mock-transfected cells. The insolubility of claudin-1 and -3 in Triton X-100 was increased by claudin-4 overexpression, but that of claudin-2, occludin, ZO-1, and E-cadherin was unchanged. Immunocytochemistry showed that claudin-4 overexpression increases the accumulation of claudin-1 and -3 in tight junctions (TJs). Furthermore, claudin-4 overexpression increased the association of claudin-4 with claudin-1 and -3. These results suggest that claudin-4 accumulates claudin-1 and -3 in TJs to enhance cell-cell contact in renal tubular epithelial cells.     

Tight Junction Proteins in Human Schwann Cell Autotypic Junctions

Tight junctions (TJs) form physical barriers in various tissues and regulate paracellular transport of ions, water, and molecules. Myelinating Schwann cells form highly organized structures, including compact myelin, nodes of Ranvier, paranodal regions, Schmidt-Lanterman incisures, periaxonal cytoplasmic collars, and mesaxons. Autotypic TJs are formed in non-compacted myelin compartments between adjacent membrane lamellae of the same Schwann cell. Using indirect immunofluorescence and RT-PCR, we analyzed the expression of adherens junction (E-cadherin) and TJ [claudins, zonula occludens (ZO)-1, occludin] components in human peripheral nerve endoneurium, showing clear differences with published rodent profiles. Adult nerve paranodal regions contained E-cadherin, claudin-1, claudin-2, and ZO-1. Schmidt-Lanterman incisures contained E-cadherin, claudin-1, claudin-2, claudin-3, claudin-5, ZO-1, and occludin. Mesaxons contained E-cadherin, claudin-1, claudin-2, claudin-3, ZO-1, and occludin. None of the proteins studied were associated with nodal inter-Schwann cell junctions. Fetal nerve expression of claudin-1, claudin-3, ZO-1, and occludin was predominantly punctate, with a mesaxonal labeling pattern, but paranodal (ZO-1, claudin-3) and Schmidt-Lanterman incisure (claudins-1 and -3) expression profiles typical of compact myelin were visible by gestational week 37. The clear differences observed between human and published rodent nerve profiles emphasize the importance of human studies when translating the results of animal models to human diseases. (J Histochem Cytochem 57:523–529, 2009)     

L-Glutamine ameliorates acetaldehyde-induced increase in paracellular permeability in Caco-2 cell monolayer

Role of L-glutamine in the protection of intestinal epithelium from acetaldehyde-induced disruption of barrier function was evaluated in Caco-2 cell monolayer. L-Glutamine reduced the acetaldehyde-induced decrease in transepithelilal electrical resistance and increase in permeability to inulin and lipopolysaccharide in a time- and dose-dependent manner; d-glutamine, L-aspargine, L-arginine, L-lysine, or L-alanine produced no significant protection. The glutaminase inhibitor 6-diazo-5-oxo-L-norleucine failed to affect the L-glutamine-mediated protection of barrier function. L-Glutamine reduced the acetaldehyde-induced redistribution of occludin, zonula occludens-1 (ZO-1), E-cadherin, and beta-catenin from the intercellular junctions. Acetaldehyde dissociates occludin, ZO-1, E-cadherin, and beta-catenin from the actin cytoskeleton, and this effect was reduced by L-glutamine. L-Glutamine induced a rapid increase in the tyrosine phosphorylation of EGF receptor, and the protective effect of L-glutamine was prevented by AG1478, the EGF-receptor tyrosine kinase inhibitor. These results indicate that L-glutamine prevents acetaldehyde-induced disruption of the tight junction and increase in the paracellular permeability in Caco-2 cell monolayer by an EGF receptor-dependent mechanism.     

Distribution of junction- and differentiation-related proteins in urothelial cells at the leading edge of primary explant outgrowths

Leading edge cells, which are located at the forefront of a wound margin, play a significant role in coordinating the wound healing process. In this study, leading edge cells of the urothelial explant outgrowth, resembling leading edge cells during urothelial full-thickness wound healing in vivo, were analyzed for expression and distribution of junction and differentiation-related proteins. Ultrastructural and immunofluorescence studies revealed that urothelial cells at the leading edge expressed ZO-1, claudin-4, occludin, E-cadherin, cytokeratin 7 and cytokeratin 20, while no expression of claudin-8 was noted. ZO-1, claudin-4, occludin and E-cadherin were localized along the cell membranes where neighbouring leading edge cells were in contact. Cytokeratin 7 was detected as filaments and cytokeratin 20 as small dots and sparse filaments. In conclusion, we detected early expression of ZO-1, claudin-4 and occludin at the urothelial leading edge, predicating the later formation of tight junctions as a necessary stage for the differentiation process that subsequently begins. The expression of occludin and cytokeratin 20 in urothelial cells at the leading edge suggests that leading edge cells may develop into fully differentiated superficial cells.     

ERK is involved in EGF-mediated protection of tight junctions, but not adherens junctions, in acetaldehyde-treated Caco-2 cell monolayers

The role of mitogen-activated protein kinases (MAPK) in the mechanism of EGF-mediated prevention of acetaldehyde-induced tight junction disruption was evaluated in Caco-2 cell monolayers. Pretreatment of cell monolayers with EGF attenuated acetaldehyde-induced decrease in resistance and increase in inulin permeability and redistribution of occludin, zona occludens-1 (ZO-1), E-cadherin, and β-catenin from the intercellular junctions. EGF rapidly increased the levels of phospho-ERK1/2, phospho-p38 MAPK, and phospho-JNK1. Pretreatment of cell monolayers with U-0126 (inhibitor of ERK activation), but not SB-202190 and SP-600125 (p38 MAPK and JNK inhibitors), significantly attenuated EGF-mediated prevention of acetaldehyde-induced changes in resistance, inulin permeability, and redistribution of occludin and ZO-1. U-0126, but not SB-202190 and SP-600125, also attenuated EGF-mediated prevention of acetaldehyde effect on the midregion F-actin ring. However, EGF-mediated preservation of junctional distribution of E-cadherin and β-catenin was unaffected by all three inhibitors. Expression of wild-type or constitutively active MEK1 attenuated acetaldehyde-induced redistribution of occludin and ZO-1, whereas dominant-negative MEK1 prevented EGF-mediated preservation of occludin and ZO-1 in acetaldehyde-treated cells. MEK1 expression did not alter E-cadherin distribution in acetaldehyde-treated cells in the presence or absence of EGF. Furthermore, EGF attenuated acetaldehyde-induced tyrosine-phosphorylation of occludin, ZO-1, claudin-3, and E-cadherin. U-0126, but not SB-202190 and SP-600125, prevented EGF effect on tyrosine-phosphorylation of occludin and ZO-1, but not claudin-3, E-cadherin, or β-catenin. These results indicate that EGF-mediated protection of tight junctions from acetaldehyde requires the activity of ERK1/2, but not p38 MAPK or JNK1/2, and that EGF-mediated protection of adherens junctions is independent of MAPK activities.     

Picroside III,an Active Ingredient of Picrorhiza scrophulariiflora,Ameliorates Experimental Colitis by Protecting Intestinal Barrier Integrity

This study aims to explore the protective effects of Picroside III, an active ingredient of Picrorhiza scrophulariiflora, on the intestinal epithelial barrier in tumor necrosis factor-α (TNF-α) induced Caco-2 cells and dextran sulfate sodium (DSS) induced colitis in mice. Results show that Picroside III significantly alleviated clinical signs of colitis including body weight loss, disease activity index increase, colon shortening, and colon tissue damage. It also increased claudin-3, ZO-1 and occludin expressions and decreased claudin-2 expression in the colon tissues of mice with colitis. In vitro, Picroside III also significantly promoted wound healing, decreased the permeability of cell monolayer, upregulated the expressions of claudin-3, ZO-1 and occludin and downregulated the expression of claudin-2 in TNF-α treated Caco-2 cells. Mechanism studies show that Picroside III significantly promoted AMP-activated protein kinase (AMPK) phosphorylation in vitro and in vivo, and blockade with AMPK could significantly attenuate the upregulation of Picroside III in ZO-1 and occludin expressions and the downregulation of claudin-2 expression in TNF-α treated Caco-2 cells. In conclusion, this study demonstrates that Picroside III attenuated DSS-induced colitis by promoting colonic mucosal wound healing and epithelial barrier function recovery via the activation of AMPK.     

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.     

Oncogenic Raf-1 disrupts epithelial tight junctions via downregulation of occludin

Occludin is an integral membrane protein of the epithelial cell tight junction (TJ). Its potential role in coordinating structural and functional events of TJ formation has been suggested recently. Using a rat salivary gland epithelial cell line (Pa-4) as a model system, we have demonstrated that occludin not only is a critical component of functional TJs but also controls the phenotypic changes associated with epithelium oncogenesis. Transfection of an oncogenic Raf-1 into Pa-4 cells resulted in a complete loss of TJ function and the acquisition of a stratified phenotype that lacked cell-cell contact growth control. The expression of occludin and claudin-1 was downregulated, and the distribution patterns of ZO-1 and E-cadherin were altered. Introduction of the human occludin gene into Raf-1-activated Pa-4 cells resulted in reacquisition of a monolayer phenotype and the formation of functionally intact TJs. In addition, the presence of exogenous occludin protein led to a recovery in claudin-1 protein level, relocation of the zonula occludens 1 protein (ZO-1) to the TJ, and redistribution of E-cadherin to the lateral membrane. Furthermore, the expression of occludin inhibited anchorage-independent growth of Raf-1-activated Pa-4 cells in soft agarose. Thus, occludin may act as a pivotal signaling molecule in oncogenic Raf- 1-induced disruption of TJs, and regulates phenotypic changes associated with epithelial cell transformation.     

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.     

Occludin S408 phosphorylation regulates tight junction protein interactions and barrier function

Although the C-terminal cytoplasmic tail of the tight junction protein occludin is heavily phosphorylated, the functional impact of most individual sites is undefined. Here, we show that inhibition of CK2-mediated occludin S408 phosphorylation elevates transepithelial resistance by reducing paracellular cation flux. This regulation requires occludin, claudin-1, claudin-2, and ZO-1. S408 dephosphorylation reduces occludin exchange, but increases exchange of ZO-1, claudin-1, and claudin-2, thereby causing the mobile fractions of these proteins to converge. Claudin-4 exchange is not affected. ZO-1 domains that mediate interactions with occludin and claudins are required for increases in claudin-2 exchange, suggesting assembly of a phosphorylation-sensitive protein complex. Consistent with this, binding of claudin-1 and claudin-2, but not claudin-4, to S408A occludin tail is increased relative to S408D. Finally, CK2 inhibition reversed IL-13-induced, claudin-2-dependent barrier loss. Thus, occludin S408 dephosphorylation regulates paracellular permeability by remodeling tight junction protein dynamic behavior and intermolecular interactions between occludin, ZO-1, and select claudins, and may have therapeutic potential in inflammation-associated barrier dysfunction.     

Epidermal growth factor receptor activation differentially regulates claudin expression and enhances transepithelial resistance in Madin-Darby canine kidney cells

Tight junctions (TJs) are the most apical cell-cell junctions, and claudins, the recently identified TJ proteins, are critical for maintaining cell-cell adhesion in epithelial cell sheets. Based on their in vivo distribution and the results of overexpression studies, certain claudins, including claudin-1 and -4, are postulated to increase, whereas other claudins, especially claudin-2, are postulated to decrease the overall transcellular resistance. The overall ratio among claudins expressed in a cell/tissue has been hypothesized to define the complexity of TJs. Disruption of the TJs contributes to various human diseases, and a correlation between reduction of TJ function and tumor dedifferentiation has been postulated. The epidermal growth factor (EGF) receptor (EGFR) is overexpressed in a wide spectrum of epithelial cancers, and its expression correlates with a more metastatic cancer phenotype. However, normal functioning of EGFR is essential for normal epithelial cell proliferation and differentiation. The role of EGFR-dependent signaling in the development and maintenance of epithelial TJ integrity has not been studied in detail. This study demonstrates that, in polarized Madin-Darby canine kidney II cells, EGF-induced EGFR activation significantly inhibited claudin-2 expression while simultaneously inducing cellular redistribution and increased expression of claudin-1, -3, and -4. Accompanying these EGF-induced changes in claudin expression was a 3-fold increase in transepithelial resistance, a functional measure of TJs. In contrast, there were no alterations in protein expression and/or intracellular localization of other TJ-related proteins (ZO-1 and occludin) or adherens junction-associated proteins (E-cadherin and beta-catenin), suggesting that EGF regulates TJ function through selective and differential regulation of claudins.     

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.     

Protein phosphatase 2A associates with and regulates atypical PKC and the epithelial tight junction complex

Tight junctions (TJs) play a crucial role in the establishment of cell polarity and regulation of paracellular permeability in epithelia. Here, we show that upon calcium-induced junction biogenesis in Madin-Darby canine kidney cells, ABalphaC, a major protein phosphatase (PP)2A holoenzyme, is recruited to the apical membrane where it interacts with the TJ complex. Enhanced PP2A activity induces dephosphorylation of the TJ proteins, ZO-1, occludin, and claudin-1, and is associated with increased paracellular permeability. Expression of PP2A catalytic subunit severely prevents TJ assembly. Conversely, inhibition of PP2A by okadaic acid promotes the phosphorylation and recruitment of ZO-1, occludin, and claudin-1 to the TJ during junctional biogenesis. PP2A negatively regulates TJ assembly without appreciably affecting the organization of F-actin and E-cadherin. Significantly, inhibition of atypical PKC (aPKC) blocks the calcium- and serum-independent membrane redistribution of TJ proteins induced by okadaic acid. Indeed, PP2A associates with and critically regulates the activity and distribution of aPKC during TJ formation. Thus, we provide the first evidence for calcium-dependent targeting of PP2A in epithelial cells, we identify PP2A as the first serine/threonine phosphatase associated with the multiprotein TJ complex, and we unveil a novel role for PP2A in the regulation of epithelial aPKC and TJ assembly and function.