Neutrophil migration across a cultured epithelial monolayer elicits a biphasic resistance response representing sequential effects on transcellular and paracellular pathways

Migration of polymorphonuclear leukocytes across epithelia is a hallmark of many inflammatory disease states. Neutrophils traverse epithelia by migrating through the paracellular space and crossing intercellular tight junctions. We have previously shown (Nash, S., J. Stafford, and J.L. Madara. 1987. J. Clin. Invest. 80:1104-1113), that leukocyte migration across T84 monolayers, a model human intestinal epithelium, results in enhanced tight junction permeability--an effect quantitated by the use of a simple, standard electrical assay of transepithelial resistance. Here we show that detailed time course studies of the transmigration-elicited decline in resistance has two components, one of which is unrelated to junctional permeability. The initial decrease in resistance, maximal 5-13 min after initiation of transmigration, occurs despite inhibition of transmigration by an antibody to the common beta subunit of neutrophil beta 2 integrins, and is paralleled by an increase in transepithelial short-circuit current. Chloride ion substitution and inhibitor studies indicate that the early-phase resistance decline is not attributable to an increase in tight junction permeability but is due to decreased resistance across epithelial cells resulting from chloride secretion. Since T84 cells are accepted models for studies of the regulation of Cl- and water secretion, our results suggest that neutrophil transmigration across mucosal surfaces (for example, respiratory and intestinal tracts) may initially activate flushing of the surface by salt and water. Equally important, these studies, by providing a concrete example of sequential transcellular and paracellular effects on transepithelial resistance, highlight the fact that this widely used assay cannot simply be viewed as a direct functional probe of tight junction permeability.     

A Synthetic Peptide Corresponding to the Extracellular Domain of Occludin Perturbs the Tight Junction Permeability Barrier

Occludin, the putative tight junction integral membrane protein, is an attractive candidate for a protein that forms the actual sealing element of the tight junction. To study the role of occludin in the formation of the tight junction seal, synthetic peptides (OCC1 and OCC2) corresponding to the two putative extracellular domains of occludin were assayed for their ability to alter tight junctions in Xenopus kidney epithelial cell line A6. Transepithelial electrical resistance and paracellular tracer flux measurements indicated that the second extracellular domain peptide (OCC2) reversibly disrupted the transepithelial permeability barrier at concentrations of < 5 μM. Despite the increased paracellular permeability, there were no changes in gross epithelial cell morphology as determined by scanning EM. The OCC2 peptide decreased the amount of occludin present at the tight junction, as assessed by indirect immunofluorescence, as well as decreased total cellular content of occludin, as assessed by Western blot analysis. Pulse-labeling and metabolic chase analysis suggested that this decrease in occludin level could be attributed to an increase in turnover of cellular occludin rather than a decrease in occludin synthesis. The effect on occludin was specific because other tight junction components, ZO-1, ZO-2, cingulin, and the adherens junction protein E-cadherin, were unaltered by OCC2 treatment. Therefore, the peptide corresponding to the second extracellular domain of occludin perturbs the tight junction permeability barrier in a very specific manner. The correlation between a decrease in occludin levels and the perturbation of the tight junction permeability barrier provides evidence for a role of occludin in the formation of the tight junction seal.     

Opposite regulation of transepithelial electrical resistance and paracellular permeability by Rho in Madin-Darby canine kidney cells.

Small GTPase Rho has been thought to be important for the formation and the maintenance of tight junction in epithelial cells, but the role of Rho in the regulation of barrier function of tight junction is not well understood. We here examined whether Rho was involved in the barrier function of tight junction in Madin-Darby canine kidney (MDCK) cells. The activation of prostaglandin EP3beta receptor, coupled to a Rho activation pathway, induced the increase in transepithelial electrical resistance (TER) but the increase in paracellular flux of mannitol in the preformed monolayer of the MDCK cells expressing the EP3beta receptor. This effect of the EP3 receptor was mimicked by the expression of constitutively active RhoA but not by active Rac1 in MDCK cells, using an isopropyl-beta-D-thiogalactoside-inducible expression system. On the other hand, the activation of EP3beta receptor suppressed the elevation of TER and the decrease in paracellular mannitol flux during Ca(2+) switch-induced tight junction formation, whereas the expression of active RhoA or Rac1 did not apparently affect the TER development in the Ca(2+) switch. These results demonstrate that the EP3 receptor and active RhoA regulate permeabilities of ionic and nonionic molecules in opposite directions in the preformed monolayer, and the EP3 receptor suppresses the elevation of TER during the tight junction formation.     

Entamoeba histolytica disturbs the tight junction complex in human enteric T84 cell layers.

Entamoeba (E.) histolytica trophozoites initiate amebiasis through invasion into the enteric mucosa. It was our aim to understand the molecular interactions between amebic trophozoites and enterocytes during the early steps of invasion. Trophozoites of E. histolytica strain HM1:IMSS were seeded on the apical side of enteric T84 cell layers, which were established on filters in two-compartment culture chambers. Cocultures were analyzed for paracellular permeability by measurement of transepithelial electrical resistance (TER) and for the tight junction proteins ZO-1, ZO-2, occludin, and cingulin by immunocytochemistry and immunoprecipitation. On direct contact with the apical side of the enteric cells, trophozoites caused an increase in paracellular permeability as evidenced by a decrease of TER associated with an increase in [(3)H]mannitol flux. Immunoprecipitation of cocultures revealed dephosphorylation of ZO-2, loss of ZO-1 from ZO-2, and degradation of ZO-1 but less so of ZO-2 and none of occludin or E-cadherin. In conclusion, trophozoite-associated increase in paracellular permeability of enteric cell layers is ascribed to disturbance of the molecular organization of tight junction proteins.     

Microtubule Integrity Is Necessary for the Epithelial Barrier Function of Cultured Thyroid Cell Monolayers

Vectorial transport in the thyroid epithelium requires an efficient barrier against passive paracellular flux, a role which is principally performed by the tight junction (zonula occludens). There is increasing evidence that tight junction integrity is determined by integral and peripheral membrane proteins which interact with the cell cytoskeleton. Although the contribution of the actin cytoskeleton to tight junction physiology has been intensively studied, less is known about possible interactions with microtubules. In the present study we used electrophysiological and immunohistochemical approaches to investigate the contribution of microtubules to the paracellular barrier in cultured thyroid cell monolayers which displayed a high transepithelial electrical resistance (6000-9000 ohm · cm²). Colchicine (1 μM) caused a progressive fall in electrical resistance to <10% of baseline after 6 h and depolarization of the transepithelial electrical potential difference consistent with a significant increase in paracellular permeability. The effect of colchicine on TER was not affected by agents which inhibit the major apical conductances of thyroid cells but was reversed upon removal of the drug. Immunofluorescent staining for tubulin combined with confocal laser scanning microscopy demonstrated that thyroid cells possessed a dense microtubule network extending throughout the cytoplasm which was destroyed by colchicine. Colchicine also produced changes in the localization of the tight junction-associated protein, ZO-1: its normally continuous junctional distribution was disrupted by striking discontinuities and the appearance of many fine strands which extended into the cytoplasm. A similar disruption in E-cadherin staining was also observed, but colchicine did not affect the distribution of vinculin associated with adherens junctions nor the integrity of the perijunctional actin ring. We conclude that microtubules are necessary for the functional and structural integrity of tight junctions in this electrically tight, transporting epithelium.     

Effect of FCCP on tight junction permeability and cellular distribution of ZO-1 protein in epithelial (MDCK) cells

The effect of the uncoupler of oxidative phosphorylation, FCCP (carbonylcyanide p-trifluoromethoxyphenylhydrazone), on the tight junction of Madin-Darby canine kidney cells was examined. FCCP induced an abrupt decrease in the transepithelial electrical resistance of the confluent monolayers over a period of 20 s. When FCCP was withdrawn from the incubation medium, the monolayer resistance recovered to close to the original level in less than 2 h. Staining of the tight junction-associated protein ZO-1 showed that the changes in transepithelial electrical resistance were accompanied by a diffusing of the protein away from cell peripheries and a reconcentration to the tight junction areas following resistance recovery. Intracellular pH was decreased by FCCP on a similar time-scale with no obvious changes in ATP levels over this time-course. These data suggest that the uncoupler FCCP has a profound effect on tight junction permeability and cellular distribution of the tight junction protein ZO-1 in the epithelial cells and that it probably acts by breaking down proton gradients and altering intracellular pH.     

Roles of Rho/ROCK and MLCK in TNF-alpha-induced changes in endothelial morphology and permeability

Tumor necrosis factor-alpha (TNF-alpha) is known to induce changes in endothelial cell morphology and permeability, but the mechanisms have not been extensively characterized. TNF-alpha rapidly induced RhoA activation and myosin light chain phosphorylation, but caused only small changes to cortical F-actin, without significantly increasing paracellular permeability up to 30 min after stimulation. TNF-alpha subsequently caused a progressive increase in permeability and in stress fiber reorganization, cell elongation, and intercellular gap formation over 8-24 h. Consistent with the increased permeability, Occludin and JAM-A were removed from tight junctions and ZO-1 was partially redistributed. Rho/ROCK but not MLCK inhibition prevented the long-term TNF-alpha-induced changes in F-actin and cell morphology, but ROCK inhibition did not affect permeability. These results suggest that the gradual increase in permeability induced by TNF-alpha does not reflect contractile mechanisms mediated by Rho, ROCK, and MLCK, but involves long-term reorganization of tight junction proteins.     

Tight junction structure and ZO-1 content are identical in two strains of Madin-Darby canine kidney cells which differ in transepithelial resistance

The relationship of tight junction permeability to junction structure and composition was examined using two strains of Madin-Darby canine kidney (MDCK) cells (I and II) which differ greater than 30-fold in transepithelial resistance. This parameter is largely determined by paracellular, and hence junctional, permeability under most conditions. When these two strains of cells were grown on permeable filter supports, they formed monolayers with equivalent linear amounts of junction/area of monolayer. Ultrastructural analysis of these monolayers by thin section EM revealed no differences in overall cellular morphology or in tight junction organization. Morphometric analysis of freeze-fractured preparations indicated that the tight junctions of these two cell strains were similar in both number and density of junctional fibrils. Prediction of transepithelial resistance for the two strains from this freeze-fracture data and a published structure-function formulation (Claude, P. 1978, J. Memb. Biol. 39:219- 232) yielded values (I = 26.5 omega/cm2, II = 35.7 omega/cm2) that were significantly lower than those observed (I = 2,500-5,000 omega/cm2, II = 50-70 omega/cm2). Consistent with these structural studies, a comparison of the distribution and cellular content of ZO-1, a polypeptide localized exclusively to the tight junction, revealed no significant differences in either the localization of ZO-1 or the amount of ZO-1 per micron of junction (I = 1,415 +/- 101 molecules/micron, II = 1,514 +/- 215 molecules/micron).     

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.     

Occludin modulates transepithelial migration of neutrophils

Neutrophils cross epithelial sheets to reach inflamed mucosal surfaces by migrating along the paracellular route. To avoid breakdown of the epithelial barrier, this process requires coordinated opening and closing of tight junctions, the most apical intercellular junctions in epithelia. To determine the function of epithelial tight junction proteins in this process, we analyzed neutrophil migration across monolayers formed by stably transfected epithelial cells expressing wild-type and mutant occludin, a membrane protein of tight junctions with four transmembrane domains and both termini in the cytosol. We found that expression of mutants with a modified N-terminal cytoplasmic domain up-regulated migration, whereas deletion of the C-terminal cytoplasmic domain did not have an effect. The N-terminal cytosolic domain was also found to be important for the linear arrangement of occludin within tight junctions but not for the permeability barrier. Moreover, expression of mutant occludin bearing a mutation in one of the two extracellular domains inhibited neutrophil migration. The effects of transfected occludin mutants on neutrophil migration did not correlate with their effects on selective paracellular permeability and transepithelial electrical resistance. Hence, specific domains and functional properties of occludin modulate transepithelial migration of neutrophils.     

Truncation mutants of the tight junction protein ZO-1 disrupt corneal epithelial cell morphology

The tight junction is the most apical intercellular junction of epithelial cells and regulates transepithelial permeability through the paracellular pathway. To examine possible functions for the tight junction-associated protein ZO-1, C-terminally truncated mutants and a deletion mutant of ZO-1 were epitope tagged and stably expressed in corneal epithelial cell lines. Only full-length ZO-1 and one N-terminal truncation mutant targeted to cell borders; other mutants showed variable cytoplasmic distributions. None of the mutants initially disrupted the localization of endogenous ZO-1. However, long-term stable expression of two of the N-terminal mutants resulted in a dramatic change in cell shape and patterns of gene expression. An elongated fibroblast-like shape replaced characteristic epithelial cobblestone morphology. In addition, vimentin and smooth muscle actin expression were up-regulated, although variable cytokeratin expression remained, suggesting a partial transformation to a mesenchymal cell type. Concomitant with the morphological change, the expression of the integral membrane tight junction protein occludin was significantly down-regulated. The localizations of endogenous ZO-1 and another family member, ZO-2, were disrupted. These findings suggest that ZO-1 may participate in regulation of cellular differentiation.     

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

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

Simvastatin Alleviates Hyperpermeability of Glomerular Endothelial Cells in Early-Stage Diabetic Nephropathy by Inhibition of RhoA/ROCK1

Background

Endothelial dysfunction is an early sign of diabetic cardiovascular disease and may contribute to progressive diabetic nephropathy (DN). There is increasing evidence that dysfunction of the endothelial tight junction is a crucial step in the development of endothelial hyperpermeability, but it is unknown whether this occurs in glomerular endothelial cells (GEnCs) during the progression of DN. We examined tight junction dysfunction of GEnCs during early-stage DN and the potential underlying mechanisms. We also examined the effect of simvastatin (3-Hydroxy-3-methylglutaryl CoA reductase inhibitor) on dysfunction of the tight junctions of cultured GEnCs and in db/db mice with early-stage DN.

Methods

We assessed the expression of occludin and ZO-1, two major components of the tight junction complex, in cultured rat GEnCs treated with high glucose and in 12 week-old db/db mice with early-stage DN. We also investigated activation of RhoA/ROCK1 signaling, GEnC permeability, and renal function of the mice.

Results

High glucose suppresses occludin expression and disrupts occludin/ZO-1 translocation in GEnCs. These changes were associated with increased permeability to albumin and activation of RhoA/ROCK1 signaling. Occludin and ZO-1 dysregulation also occurred in the glomeruli of mice with early-stage DN, and these abnormalities were accompanied by albuminuria and activation of RhoA/ROCK1 in isolated glomeruli. Simvastatin prevented high glucose or hyperglycemia-induced dysregulation of occludin and ZO-1 by inhibition of RhoA/ROCK1 signaling in cultured GEnCs and in db/db mice with early-stage DN.

Conclusion

Our results indicate that activation of RhoA/ROCK1 by high glucose disrupts the expression and translocation of occludin/ZO-1 and that simvastatin alleviates occludin/ZO-1 dysregulation and albuminuria by suppressing RhoA/ROCK1 signaling during early-stage DN. These results suggest a potential therapeutic strategy for preventing the onset of albuminuria in early-stage DN.     

Pseudomonas aeruginosa quorum sensing molecule N-(3 oxododecanoyl)-l-homoserine lactone disrupts epithelial barrier integrity of Caco-2 cells

Acyl-homoserine lactone (HSL) quorum sensing molecules play an important role in regulation of virulence gene expression in Pseudomonas aeruginosa. Here, we show that 3O-C(12)-HSL can disrupt barrier integrity in human epithelial Caco-2 cells as evidenced by decreased transepithelial electrical resistance (TER), increased paracellular flux, reduction in the expression and distribution of ZO-1 and occludin, and reorganization of F-actin. P. aeruginosa 3O-C(12)-HSL activate p38 and p42/44 kinases, and inhibition of these kinases partly prevented 3O-C(12)-HSL-induced changes in TER, paracellular flux and expression of occludin and ZO-1. These findings demonstrate that P. aeruginosa 3O-C(12)-HSL can modulate tight junction integrity of Caco-2 cells.     

Role of phospholipase Cgamma-induced activation of protein kinase Cepsilon (PKCepsilon) and PKCbetaI in epidermal growth factor-mediated protection of tight junctions from acetaldehyde in Caco-2 cell monolayers

Epidermal growth factor (EGF) protects the intestinal epithelial tight junctions from acetaldehyde-induced insult. The role of phospholipase Cgamma (PLCgamma) and protein kinase C (PKC) isoforms in the mechanism of EGF-mediated protection of tight junction from acetaldehyde was evaluated in Caco-2 cell monolayers. EGF-mediated prevention of acetaldehyde-induced decrease in transepithelial electrical resistance and an increase in inulin permeability, and subcellular redistribution of occludin and ZO-1 was attenuated by reduced expression of PLCgamma1 by short hairpin RNA. EGF induced a rapid activation of PLCgamma1 and PLC-dependent membrane translocation of PKCepsilon and PKCbetaI. Inhibition of PKC activity or selective interference of membrane translocation of PKCepsilon and PKCbetaI by RACK interference peptides attenuated EGF-mediated prevention of acetaldehyde-induced increase in inulin permeability and redistribution of occludin and ZO-1. BAPTA-AM and thapsigargin blocked EGF-induced membrane translocation of PKCbetaI and attenuated EGF-mediated prevention of acetaldehyde-induced disruption of tight junctions. EGF-induced translocation of PKCepsilon and PKCbetaI was associated with organization of F-actin near the perijunctional region. This study shows that PLCgamma-mediated activation of PKCepsilon and PKCbetaI and intracellular calcium is involved in EGF-mediated protection of tight junctions from acetaldehyde-induced insult.     

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.     

ZO-2 silencing in epithelial cells perturbs the gate and fence function of tight junctions and leads to an atypical monolayer architecture

ZO-2 is a tight junction (TJ) protein that shuttles between the plasma membrane and the nucleus. ZO-2 contains several protein binding sites that allow it to function as a scaffold that clusters integral, adaptor and signaling proteins. To gain insight into the role of ZO-2 in epithelial cells, ZO-2 was silenced in MDCK cells with small interference RNA (siRNA). ZO-2 silencing triggered: (A) changes in the gate function of the TJ, determined by an increase in dextran flow through the paracellular route of mature monolayers and achievement of lower transepithelial electrical resistance values upon TJ de novo formation; (B) changes in the fence function of the TJ manifested by a non-polarized distribution of E-cadherin on the plasma membrane; (C) altered expression of TJ and adherens junction proteins, determined by a decreased amount of occludin and E-cadherin in mature monolayers and a delayed arrival to the plasma membrane of ZO-1, occludin and E-cadherin during a calcium switch assay; and (D) an atypical monolayer architecture characterized by the appearance of widened intercellular spaces, multistratification of regions in the culture and an altered pattern of actin at the cellular borders.     

Phospholipase C-gamma modulates epithelial tight junction permeability through hyperphosphorylation of tight junction proteins

Phospholipase C-gamma (PLC-gamma) is stimulated by epidermal growth factor via activation of the epidermal growth factor receptors. The PLC inhibitor, 3-nitrocoumarin (3-NC), selectively inhibited PLC-gamma in Madin-Darby canine kidney cells without affecting the activity of PLC-beta. In contrast, inhibitors of PLC-beta, hexadecylphosphocholine and, had no effect on the activity of PLC-gamma. Inhibition of PLC-gamma by 3-NC was associated with an increase in tight junction permeability across Madin-Darby canine kidney cell monolayers, as evidenced by 3-NC-induced decrease in transepithelial electrical resistance and increase in mannitol flux over a concentration range that was inhibitory to PLC-gamma. An analog of 3-NC, 7-hydroxy-3-NC (7-OH-3-NC), which was inactive as an inhibitor of PLC-gamma, also had no effect on tight junction permeability. Treatment with 3-NC caused punctate disruption in the cortical actin filaments. The PLC-gamma inhibitor, 3-NC, but not the inactive analog, 7-OH-3-NC, caused hyperphosphorylation of the tight junction proteins, occludin, ZO-1, and ZO-2. The serine/threonine kinase inhibitor, staurosporine (50-200 nm), significantly attenuated 3-NC-induced hyperphosphorylation of ZO-2. This corresponded with attenuation by staurosporine of 3-NC-induced increase in tight junction permeability, suggesting a relationship between ZO-2 phosphorylation and tight junction permeability.     

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.     

Role of phosphatidylinositol 3-kinase in oxidative stress-induced disruption of tight junctions

A recent study (Nusrat, A., Chen, J. A., Foley, C. S., Liang, T. W., Tom, J., Cromwell, M., Quan, C., and Mrsny, R. J. (2000) J. Biol. Chem. 275, 29816-29822) suggested that phosphatidylinositol 3-kinase (PI 3-kinase) may interact with occludin; however, there exists no evidence of direct interaction of PI 3-kinase with the tight junctions. Activation of PI 3-kinase by oxidative stress and its role in disruption of tight junctions was examined in Caco-2 cell monolayer. The oxidative stress-induced decrease in electrical resistance, increase in inulin permeability, and redistribution of occludin and ZO-1 were reduced by a PI 3-kinase inhibitor, LY294002. Oxidative stress-induced tyrosine phosphorylation and dissociation from the actin cytoskeleton of occludin and ZO-1 were reduced by LY294002. The regulatory subunit of PI 3-kinase, p85, and the PI 3-kinase activity were co-immunoprecipitated with occludin, which were rapidly increased by oxidative stress. Oxidative stress resulted in increased translocation of p85 from the intracellular compartment into the intercellular junctions. Pair-wise glutathione S-transferase pull-down assay showed that glutathione S-transferase-occludin (C-terminal tail) binds to recombinant p85. This study shows that oxidative stress increases the association of PI 3-kinase with the occludin, and that PI 3-kinase activity is involved in oxidative stress-induced disruption of tight junction.