Platelet-derived growth factor mediates tight junction redistribution and increases permeability in MDCK cells

Increased tissue permeability is a common characteristic of a number of diseases such as pulmonary edema, inflammatory bowel disease, several kidney diseases, diabetic retinopathy, and tumors. We hypothesized that growth factors increase permeability by redistribution of tight junction proteins away from the cell border. To investigate mechanisms of growth factor-mediated permeability, we examined the effect of platelet derived growth factor (PDGF) on Madin-Darby canine kidney (MDCK) cell tight junction protein distribution and on permeability. PDGF altered the cellular distribution of occludin and ZO-1 from the cell border to the cytoplasm and increased permeability to 70 kDa dextran in a concentration-dependent manner. Treatment of MDCK cells with PDGF prior to fixation allowed binding of the lectin concanavalin A to the basement membrane of fixed cells, while binding was prevented in untreated control monolayers, implying that PDGF induced the formation of a paracellular transport pathway. Cell fractionation experiments with PDGF-treated cells revealed a novel occludin-containing low-density, detergent resistant subcellular structure, which increased in the buoyant fractions relative to occludin in the pellet in a time- and concentration-dependent manner. Immunocytochemistry revealed that a pool of internalized occludin co-labels with the early endosome marker, EEA1, suggesting that PDGF may stimulate occludin to enter an endosomal pathway. PDGF may act as a permeabilizing agent by moving tight junction proteins away from the cell border in discrete microdomains, and the effects of PDGF on permeability and tight junction protein distribution may model the regulation of epithelial and endothelial barrier properties by other peptide growth factors.     

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.     

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.     

Immunolocalization of occludin and claudin-1 to tight junctions in intact CNS vessels of mammalian retina

The distributions of occludin and claudin-1, two tight junction–associated integral membrane proteins were investigated by immunohistochemical analysis of whole-mount preparations of the blood vessels in the myelinated streak of the rabbit retina. Light microscopy revealed that occludin and claudin-1 immunoreactivities were abundant along the interface of adjacent endothelial cells of all blood vessels. Electron microscopy revealed that both proteins were distributed in a regular pattern (at regular intervals of approximately 80 nm) along the length of tight junctions, probably in the regions of tight junction strands. No other structures or cell types expressed either of these two proteins in the myelinated streak. Whereas occludin immunoreactivity was concentrated only at the tight junction interface, claudin-1 immunoreactivity also extended into the cytoplasm of the endothelial cells, suggesting a different structural role for claudin-1 than for occludin at tight junctions. Retinal pigment epithelial cells expressed occludin around their entire circumference, consistent with the function of these cells as a barrier separating the retina from the leaky vessels of the choroid. Also consistent with the association of occludin expression with vessels that exhibit functional tight junctions, this protein was expressed at only a low level in, and showed an irregular distribution along, the vessels of the choroid, a vascular bed that lacks blood-barrier properties. Further, the distribution of occludin was examined during formation and remodelling of the rat retinal vasculature. Occludin expression was evident at the leading edge of vessel formation and was found on all vessels in both the inner and outer vascular plexus. Numerous vascular segments at the early stage of vascular formation and regression lost occludin expression. The biological significance of this transient loss of occludin expression in terms of barrier function remains to be elucidated.     

Regulation of tight junction permeability and occludin phosphorylation by Rhoa-p160ROCK-dependent and -independent mechanisms

In epithelial and endothelial cells, tight junctions regulate the paracellular permeability of ions and proteins. Disruption of tight junctions by inflammation is often associated with tissue edema, but regulatory mechanisms are not fully understood. Using ECV304 cells as a model system, lysophosphatidic acid and histamine were found to increase the paracellular permeability of the tracer horseradish peroxidase. Cytoskeletal changes induced by these agents included stimulation of stress fiber formation and myosin light chain phosphorylation. Additionally, occludin, a tight junction protein, was a target for signaling events triggered by lysophosphatidic acid and histamine, events that resulted in its phosphorylation. A dominant-negative mutant of RhoA, RhoA T19N, or a specific inhibitor of Rho-activated kinases, Y-27632, prevented stress fiber formation, myosin light chain phosphorylation, occludin phosphorylation, and the increase in tracer flux in response to lysophosphatidic acid. In contrast, although RhoA T19N and Y-27632 blocked the cytoskeletal events induced by histamine, they had no effect on the stimulation of occludin phosphorylation or increased tracer flux, indicating that occludin phosphorylation may regulate tight junction permeability independently of cytoskeletal events. Thus, occludin is a target for receptor-initiated signaling events regulating its phosphorylation, and this phosphorylation may be a key regulator of tight junction permeability.     

Chloroquine and Hydroxychloroquine Increase Retinal Pigment Epithelial Layer Permeability

Antimalarials chloroquine (CQ) and hydroxychloroquine (HCQ) are widely used as antiinflammatory drugs, but side effects include retinopathy and vision loss. The objective of this study was to examine the effect of CQ and HCQ on the barrier integrity of retinal pigment epithelial (RPE) cell monolayers in vitro. Permeability of ARPE‐19 cell monolayers was determined using Fluorescein isothiocyanate (FITC)‐labeled dextran. The influence of CQ and HCQ on cell death and the expression tight junction molecules was examined. CQ and HCQ significantly increased ARPE‐19 monolayer permeability after 3 and 18 h, respectively, and enhanced mRNA levels for claudin‐1 and occludin. Cytotoxicity was only observed after 18 h exposure. Thus, CQ and HCQ rapidly enhance RPE barrier permeability in vitro, independent of cytotoxicity or loss of zonula occludens‐1, claudin‐1, and occludin expression. Our findings suggest that CQ/HCQ‐induced permeability of the RPE layer may contribute to blood–retinal barrier breakdown in case of CQ/HCQ‐induced retinopathy.     

Protein kinase Cζ phosphorylates occludin and promotes assembly of epithelial tight junctions

Protein kinases play an important role in the regulation of epithelial tight junctions. In the present study, we investigated the role of PKCζ (protein kinase Cζ) in tight junction regulation in Caco-2 and MDCK (Madin-Darby canine kidney) cell monolayers. Inhibition of PKCζ by a specific PKCζ pseudosubstrate peptide results in redistribution of occludin and ZO-1 (zona occludens 1) from the intercellular junctions and disruption of barrier function without affecting cell viability. Reduced expression of PKCζ by antisense oligonucleotide or shRNA (short hairpin RNA) also results in compromised tight junction integrity. Inhibition or knockdown of PKCζ delays calcium-induced assembly of tight junctions. Tight junction disruption by PKCζ pseudosubstrate is associated with the dephosphorylation of occludin and ZO-1 on serine and threonine residues. PKCζ directly binds to the C-terminal domain of occludin and phosphorylates it on threonine residues. Thr403, Thr404, Thr424 and Thr438 in the occludin C-terminal domain are the predominant sites of PKCζ-dependent phosphorylation. A T424A or T438A mutation in full-length occludin delays its assembly into the tight junctions. Inhibition of PKCζ also induces redistribution of occludin and ZO-1 from the tight junctions and dissociates these proteins from the detergent-insoluble fractions in mouse ileum. The present study demonstrates that PKCζ phosphorylates occludin on specific threonine residues and promotes assembly of epithelial tight junctions.     

Insulin-like growth factor-1 effects on bovine retinal endothelial cell glucose transport: role of MAP kinase

In order to maintain normal metabolism, the neuroretina is completely dependent on the constant delivery of glucose across the retinal microvascular endothelial cells comprising the inner blood-retinal barrier. Glucose uptake into these cells is influenced by various stimuli, including hypoxia and growth factors. Recently, insulin-like growth factor-1 (IGF-1) was shown to enhance retinal endothelial glucose transport in a process that is dependent on protein kinase C (PKC) and phosphatidylinositol-3 kinase (PI3 kinase). In the current study, the role of mitogen-activated protein kinase (MAP kinase) in regulating IGF-1 effects on retinal endothelial cell glucose transport was investigated in a bovine retinal endothelial cell (BREC) culture model. IGF-1 (25 ng/mL) caused a rapid increase in MAP-kinase activity and ERK phosphorylation. Inhibition of MAP kinase with PD98059 (100 microm) blocked IGF-1 enhancement of 2-deoxyglucose uptake. In order to clarify the relationship between PKC, PI3 kinase and MAP kinase in IGF-1 signaling in retinal endothelial cells, the effects of selective inhibitors of MAP kinase (PD98059), PKC (GF109203X), and PI3 kinase (wortmannin, LY294002) on signal transduction by IGF-1 were studied. Inhibition of MAP kinase abolished IGF-1 stimulation of PKC but had no effect on PI3 kinase activity, whereas inhibition of either PKC and PI3 kinase had no effect on MAP kinase phosphorylation or activity in IGF-1-treated cells. Taken together, these data demonstrate that IGF-1 stimulation of BREC glucose transport requires activation of MAP kinase and that MAP kinase is upstream from PKC but is independent of PI3 kinase in mediating the actions of IGF-1 on retinal endothelial cells.     

Interleukin-2 receptor beta subunit-dependent and -independent regulation of intestinal epithelial tight junctions

Interleukin (IL)-15 is able to regulate tight junction formation in intestinal epithelial cells. However, the mechanisms that regulate the intestinal barrier function in response to IL-15 and the involved subunits of the IL-15 ligand-receptor system are unknown. We determined the IL-2Rbeta subunit and IL-15-dependent regulation of tight junction-associated proteins in the human intestinal epithelial cell line T-84. The IL-2Rbeta subunit was expressed and induced signal transduction in caveolin enriched rafts in intestinal epithelial cells. IL-15-mediated tightening of intestinal epithelial monolayers correlated with the enhanced recruitment of tight junction proteins into Triton X-100-insoluble protein fractions. IL-15-mediated up-regulation of ZO-1 and ZO-2 expression was independent of the IL-2Rbeta subunit, whereas the phosphorylation of occludin and enhanced membrane association of claudin-1 and claudin-2 by IL-15 required the presence of the IL-2Rbeta subunit. Recruitment of claudins and hyperphosphorylated occludin into tight junctions resulted in a more marked induction of tight junction formation in intestinal epithelial cells than the up-regulation of ZO-1 and ZO-2 by itself. The regulation of the intestinal epithelial barrier function by IL-15 involves IL-2Rbeta-dependent and -independent signaling pathways leading to the recruitment of claudins, hyperphosphorylated occludin, ZO-1, and ZO-2 into the tight junctional protein complex.     

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     

Occludin Localization at the Tight Junction Requires the Second Extracellular Loop

Occludin is a transmembrane protein of the tight junction with two extracellular loops. Our previous demonstration that the extracellular loops are adhesive suggested the possibility that they contribute to localizing occludin at the tight junction. To address this question, truncated forms of occludin were generated in which one or both of the extracellular loops were deleted. These constructs were expressed in both occludin-null Rat-1 fibroblasts and in MDCK epithelial cells. The patterns of sensitivity to proteinase K suggested all constructs were present on the plasma membrane and retained the normal topology. In fibroblasts, all truncated forms of occludin colocalized with ZO-1 at regions of cell-cell contact, demonstrating that even in the absence of tight junctions cytoplasmic interactions with ZOs is sufficient to cluster occludin. In MDCK cell monolayers, both full-length and occludin lacking the first extracellular loop colocalized with ZO-1 at the tight junction. In contrast, constructs lacking the second, or both, extracellular loops were absent from tight junctions and were found only on the basolateral cell surface. By freeze-fracture electron microscopic analysis, overexpression of full length occludin induced side-to-side aggregation of fibrils within the junction, while excess occludin on the lateral membrane did not form fibrils. These results suggest that the second extracellular domain is required for stable assembly of occludin in the tight junction and that occludin influences the structural organization of the paracellular barrier. Received: 26 June 2000/Revised: 25 September 2000     

Dendritic cell transmigration through brain microvessel endothelium is regulated by MIP-1alpha chemokine and matrix metalloproteinases

Dendritic cells (DCs) accumulate in the CNS during inflammatory diseases, but the exact mechanism regulating their traffic into the CNS remains to be defined. We now report that MIP-1alpha increases the transmigration of bone marrow-derived, GFP-labeled DCs across brain microvessel endothelial cell monolayers. Furthermore, occludin, an important element of endothelial tight junctions, is reorganized when DCs migrate across brain capillary endothelial cell monolayers without causing significant changes in the barrier integrity as measured by transendothelial electrical resistance. We show that DCs produce matrix metalloproteinases (MMP) -2 and -9 and GM6001, an MMP inhibitor, decreases both baseline and MIP-1alpha-induced DC transmigration. These observations suggest that DC transmigration across brain endothelial cell monolayers is partly MMP dependent. The migrated DCs express higher levels of CD40, CD80, and CD86 costimulatory molecules and induce T cell proliferation, indicating that the transmigration of DCs across brain endothelial cell monolayers contributes to the maintenance of DC Ag-presenting function. The MMP dependence of DC migration across brain endothelial cell monolayers raises the possibility that MMP blockers may decrease the initiation of T cell recruitment and neuroinflammation in the CNS.     

Tyrosine phosphorylation of VE-cadherin and claudin-5 is associated with TGF-β1-induced permeability of centrally derived vascular endothelium

Breakdown of the inner blood-retinal barrier and the blood-brain barrier is associated with changes in tight and adherens junction-associated proteins that link vascular endothelial cells. This study aimed to test the hypothesis that transforming growth factor (TGF)-β1 increases the paracellular permeability of vascular endothelial monolayers through tyrosine phosphorylation of VE-cadherin and claudin-5. Bovine retinal and human brain capillary endothelial cells were grown as monolayers on coated polycarbonate membranes. Paracellular permeability was studied by measuring the equilibration of (14)C-inulin or fluorescence-labelled dextran. Changes in VE-cadherin and claudin-5 expression were studied by immunocytochemistry (ICC) and quantified by cell-based enzyme linked immunosorbent assays (ELISA). Tyrosine phosphorylation of VE-cadherin and claudin-5 was studied by ICC, immunoprecipitation and Western blotting. We found that exposure of endothelial cells to TGF-β1 caused a dose-dependent increase in paracellular permeability as reflected by increases in the equilibration of (14)C-inulin. This effect was enhanced by the tyrosine phosphatase inhibitor orthovanadate and attenuated by the tyrosine kinase inhibitor lavendustin A. ICC and cell-based ELISA revealed that TGF-β1 induced both dose- and time-dependent decreases in VE-cadherin and claudin-5 expression. Assessment of cell viability indicated that changes in these junction-associated proteins were not due to endothelial death or injury. ICC revealed that tyrosine phosphorylation of endothelial monolayers was greatly enhanced by TGF-β1 treatment, and immunoprecipitation of cell lysates showed increased tyrosine phosphorylation of VE-cadherin and claudin-5. Our results suggest that tyrosine phosphorylation of VE-cadherin and claudin-5 is involved in the increased paracellular permeability of central nervous system-derived vascular endothelium induced by TGF-β1.     

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.     

Pigment epithelium-derived factor inhibits advanced glycation end products-induced retinal vascular permeability

Vascular hyperpermeability associated with retinal vascular leakage is known to occur in patients with diabetes, and contributes to endothelial barrier dysfunction. This study aimed to examine the effect of pigment epithelium-derived factor (PEDF) on advanced glycation end products (AGEs)-induced endothelial cell permeability. Cultured porcine retinal endothelial cell (PREC) was exposed to AGE-modified bovine serum albumin (AGE-BSA) and the endothelial cell permeability was detected by measuring the flux of rhodamine B isothiocyanate (RITC)-dextran across the PREC monolayers. We found that AGE-BSA increased the RITC-dextran flux across a PREC monolayer and PEDF blocked the solute flux induced by AGE-BSA. In order to explore the underlying signaling mechanism of PEDF on the inhibitory effect of AGE-BSA-induced permeability, we demonstrate that PEDF could inhibit the AGE-BSA-induced permeability via phosphatidylinositol 3-kinase (PI3K)/Akt pathway. AGE-BSA also increased the endothelial cell permeability by stimulating the reactive oxygen species (ROS) generation via NADPH oxidase activity and Akt phosphorylation at Ser473. PEDF decreased ROS generation in AGE-BSA-exposed endothelial cells by suppressing the NADPH oxidase activity via down regulating the phosphorylation of p22^PHOx at Thr147. This led to blockade of AGE-induction of PI3K/Akt activation in permeability. Furthermore, PEDF inhibited the AGE-BSA-induced permeability by increased expression of tight junction protein zona occludens-1(ZO-1), co-incident with an increase in barrier properties of endothelial monolayer. Together, our results indicate that PEDF could possibly act as potent anti-permeability molecule by targeting the PI3K/Akt signaling pathway by suppressing if NADPH oxidase mediated ROS generation and ZO-1 tight junction protein and it offers potential targets to inhibit the ocular related diseases such as diabetic retinopathy.     

Aspirin induces gastric epithelial barrier dysfunction by activating p38 MAPK via claudin-7

Tight junctions create a paracellular permeability barrier that is breached when nonsteroidal anti-inflammatory drugs cause gastrointestinal injury, including increased gastrointestinal permeability. However, the mechanism by which aspirin affects the function of gastric epithelial tight junctions is unknown. Thus, we examined the effect of aspirin on gastric mucosal barrier properties and tight junction organization using MKN28, a human gastric epithelial cell line that expresses claudin-3, claudin-4, claudin-7, zonula occludens (ZO)-1, and occludin, but not claudin-2 or claudin-5, as determined by immunoblot analysis and immunofluorescent staining. Aspirin (5 mM) treatment of MKN28 gastric epithelial monolayers significantly decreased transepithelial electrical resistance and increased dextran permeability. Both aspirin-mediated permeability and phosphorylation of p38 MAPK were significantly attenuated by SB-203580 (a p38 MAPK inhibitor) but not by U-0126 (a MEK1 inhibitor) or SP-600125 (a JNK inhibitor). Aspirin significantly decreased the quantity of claudin-7 protein produced by MKN28 cells but not the quantity of claudin-3, claudin-4, ZO-1, or occludin. The aspirin-induced decrease in claudin-7 protein was completely abolished by SB-203580 pretreatment. These results demonstrate, for the first time, that claudin-7 protein is important in aspirin-induced gastric barrier loss and that p38 MAPK activity mediates this epithelial barrier dysfunction. tight junction; p38 mitogen-activated protein kinase; permeability     

Occludin Phosphorylation and Ubiquitination Regulate Tight Junction Trafficking and Vascular Endothelial Growth Factor-induced Permeability

Vascular endothelial growth factor (VEGF) alters tight junctions (TJs) and promotes vascular permeability in many retinal and brain diseases. However, the molecular mechanisms of barrier regulation are poorly understood. Here we demonstrate that occludin phosphorylation and ubiquitination regulate VEGF-induced TJ protein trafficking and concomitant vascular permeability. VEGF treatment induced TJ fragmentation and occludin trafficking from the cell border to early and late endosomes, concomitant with increased occludin phosphorylation on Ser-490 and ubiquitination. Furthermore, both co-immunoprecipitation and immunocytochemistry demonstrated that VEGF treatment increased the interaction between occludin and modulators of intracellular trafficking that contain the ubiquitin interacting motif, including Epsin-1, epidermal growth factor receptor pathway substrate 15 (Eps15), and hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs). Inhibiting occludin phosphorylation by mutating Ser-490 to Ala suppressed VEGF-induced ubiquitination, inhibited trafficking of TJ proteins, and prevented the increase in endothelial permeability. In addition, an occludin-ubiquitin chimera disrupted TJs and increased permeability without VEGF. These data demonstrate a novel mechanism of VEGF-induced occludin phosphorylation and ubiquitination that contributes to TJ trafficking and subsequent vascular permeability.Under normal physiological conditions the blood-brain barrier and blood-retinal barrier regulate the transport of water, ions, amino acids, and waste products, between the neural parenchyma and blood (1). A high degree of well developed tight junctions (TJs)² in the vascular endothelium, in association with adherens junctions, contribute to both the blood-brain and blood-retinal barriers (2). Accumulating evidence suggests that a number of pathological eye diseases such as diabetes, retinopathy of prematurity, age-related macular degeneration, inflammation, and infectious diseases disrupt the TJs altering the blood-retinal barrier. Common mediators of vascular permeability and TJ deregulation are growth factors and cytokines that may induce macular edema and lead to loss of vision (1). Vascular endothelial growth factor (VEGF), in particular, induces vascular permeability and stimulates angiogenesis, contributing to disease pathogenesis in diabetic retinopathy and retinopathy of prematurity (3). VEGF also contributes to blood-brain barrier disruption with subsequent edema and angiogenesis in brain tumors and stroke (4). Recent advances in biomedical research have provided therapeutic approaches to neutralize VEGF; however, these strategies have not yet demonstrated effective resolution of diabetic macular edema (5, 6).TJs control the paracellular flux of solutes and fluids across the blood-brain and blood-retinal barriers. Several transmembrane proteins including occludin, tricellulin, the claudin family, and junction adhesion molecules are thought to confer adhesion to the TJ barrier and to be organized by members of the zonula occludens family (ZO-1, -2, or -3) (7–9). Experimental evidence has established that the claudins confer barrier properties and claudin-5 specifically contributes to the vascular component of the blood-brain barrier demonstrated by gene deletion studies (10). In contrast, the function of occludin in paracellular flux has remained less clear. Mice with occludin gene deletion continue to form TJs in gut epithelia with normal barrier properties (11). However, studies have also demonstrated that diabetes reduces occludin content in rat retina (12) and alters its distribution from continuous cell border localization to intracellular puncta (13). These observations suggest that the intracellular trafficking of TJ proteins promotes paracellular flux and vascular permeability in diabetic animals (12, 14).VEGF was originally identified as a vascular permeability factor as well as a pro-angiogenic growth factor (15, 16). Both biological effects exacerbate the pathology of retinal vascular diseases (17), and they are mediated via intracellular signal transduction, especially based on the phosphorylation of Src, protein kinase C, and so on (18). Additionally, VEGF treatment and diabetes induce occludin phosphorylation in rat retinal vasculature and endothelial cell culture coincident with increased permeability (19). Recently, using mass spectrometry five occludin phosphorylation sites were identified in retinal endothelial cell culture after VEGF treatment (20). Among these sites, phosphorylation at Ser-490 was shown to increase in response to VEGF treatment. However, no evidence has directly demonstrated the contribution of occludin phosphorylation to VEGF-induced endothelial permeability or defined the mechanism by which phosphorylation of occludin alters paracellular flux.Modification of proteins with monomeric or polymeric ubiquitin chains contributes to control of multiple biological functions including protein degradation, intracellular trafficking, translational regulation, and DNA repair (21). Phosphorylation of receptor tyrosine kinases, such as epidermal growth factor receptor or vascular endothelial growth factor receptor-2, is followed by ubiquitination and regulated trafficking to endosomes. This endocytosis process depends on the interaction between the ubiquitinated receptors and carrier proteins that possess a ubiquitin interacting motif (UIM) such as Epsin, epidermal growth factor receptor pathway substrate 15 (Eps15), and hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) (21–24). Recent publications have demonstrated that occludin can be ubiquitinated targeting the protein for degradation through the ubiquitin-proteasome system in epithelial cell types (25, 26). Here we demonstrate that phosphorylation of occludin at Ser-490 is necessary for occludin ubiquitination in response to VEGF in endothelial cells. Furthermore, the ubiquitination promotes interaction of occludin with UIM containing modulators of trafficking and regulates the internalization of TJ proteins altering endothelial permeability. Together, these results suggest that occludin phosphorylation and subsequent ubiquitination are necessary for VEGF-induced TJ trafficking and endothelial permeability.     

Platelet endothelial cell adhesion molecule‐1 (PECAM1) plays a critical role in the maintenance of human vascular endothelial barrier function

Cardiovascular endothelial barrier dysfunction is associated with a number of cardiovascular diseases. This study aims to investigate the role of platelet endothelial cell adhesion molecule‐1 (PECAM1) in the maintenance of the vascular endothelial barrier integrate. Human umbilical vein endothelial cells (HUVECs) were cultured into monolayers using as an in vitro model to assess the endothelial barrier function. Knockdown of the gene of PECAM1 markedly reduced the transendothelial resistance and increased the permeability of the HUVEC monolayers. From the wild HUVECs, we detected a complex of PECAM1, claudin1, occluding and endothelial cell selective adhesion molecule (ESAM); such a complex was not detected in the PECAM1‐deficient HUVECs. Knockdown of either claudin1, or occludin, or ESAM, did not affect the formation of the tight junction (TJ) complex. Exposure to recombinant interleukin (IL)‐13 inhibited the expression of PECAM1 and down‐regulated the HUVEC monolayer barrier function. PECAM1 plays an important role in the formation of TJ complex. Copyright © 2015 John Wiley & Sons, Ltd.     

Expression of kinase-inactive c-Src delays oxidative stress-induced disassembly and accelerates calcium-mediated reassembly of tight junctions in the Caco-2 cell monolayer

The activity of Src kinases appears to play a role in both assembly and disassembly of tight junction. However, the role of a specific isoform of Src kinase in regulation of tight junction is not known. In the present study the role of c-Src in regulation of epithelial tight junction was investigated in Caco-2 cell monolayers. Oxidative stress (xanthine oxidase + xanthine) induced an activation and membrane translocation of c-Src. The oxidative stress-induced decrease in transepithelial electrical resistance, increase in inulin permeability, and redistribution of occludin and ZO-1 from the intercellular junctions were prevented by PP2. The rates of oxidative stress-induced activation of c-Src, tyrosine phosphorylation of ZO-1 and beta-catenin, decrease in resistance, increase in permeability to inulin, and redistribution of occludin and ZO-1 were significantly greater in cells transfected with wild type c-Src, whereas it was low in cells transfected with kinase-inactive c-SrcK297R mutant, when compared with those in empty vector-transfected cells. The rates of recovery of resistance, increase in barrier to inulin, and reorganization of occludin and ZO-1 into the intercellular junctions during the calcium-induced reassembly of tight junction were much greater in Caco-2 cells transfected with c-SrcK297R as compared with those in cells transfected with empty vector or wild type c-Src. These results show that the dominant-negative expression of kinase-inactive c-Src delays the oxidative stress-induced disruption of tight junction and accelerates calcium-induced assembly of tight junction in Caco-2 cells and demonstrate that oxidative stress-induced disruption of tight junction is mediated by the activation of c-Src.     

Epithelial transport and barrier function in occludin-deficient mice

BACKGROUND AND AIMS: This study aimed at functional characterization of the tight junction protein occludin using the occludin-deficient mouse model. METHODS: Epithelial transport and barrier functions were characterized in Ussing chambers. Impedance analysis revealed the ionic permeability of the epithelium (Re, epithelial resistance). Conductance scanning differentiated transcellular (Gc) and tight junctional conductance (Gtj). The pH-stat technique quantified gastric acid secretion. RESULTS: In occludin+/+ mice, Re was 23+/-5 Omega cm2 in jejunum, 66+/-5 Omega cm2 in distal colon and 33+/-6 Omega cm2 in gastric corpus and was not altered in heterozygotic occludin+/- or homozygotic occludin-/- mice. Additionally, [3H]mannitol fluxes were unaltered. In the control colon, Gc and Gtj were 7.6+/-1.0 and 0.3+/-0.1 mS/cm2 and not different in occludin deficiency. Epithelial resistance after mechanical perturbation or EGTA exposition (low calcium switch) was not more affected in occludin-/- mice than in control. Barrier function was measured in the urinary bladder, a tight epithelium, and in the stomach. Control Rt was 5.8+/-0.8 kOmega cm2 in urinary bladder and 33+/-6 Omega cm2 in stomach and not altered in occludin-/- mice. In gastric corpus mucosa, the glandular structure exhibited a complete loss of parietal cells and mucus cell hyperplasia, as a result of which acid secretion was virtually abolished in occludin-/- mice. CONCLUSION: Epithelial barrier characterization in occludin-deficiency points against an essential barrier function of occludin within the tight junction strands or to a substitutional redundancy of single tight junction molecules like occludin. A dramatic change in gastric morphology and secretory function indicates that occludin is involved in gastric epithelial differentiation.