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.     

Role of protein tyrosine phosphorylation in acetaldehyde-induced disruption of epithelial tight junctions

Acetaldehyde-induced cytotoxicity is an important factor in pathogenesis of alcohol-related diseases; however, the mechanism of this toxicity is unknown. We recently showed that acetaldehyde increases epithelial paracellular permeability. We asked whether protein tyrosine phosphorylation via modulation of tyrosine kinases and/or PTPases is a mechanism involved in acetaldehyde-induced disruption of the tight junctions in the Caco-2 cell monolayer. Immunofluorescence localization of occludin and ZO-1 showed disruption of the tight junctions in acetaldehyde-treated cell monolayer. Administration of genistein prevented acetaldehyde-induced permeability. Acetaldehyde increased tyrosine phosphorylation of three clusters of proteins with molecular masses of 30-50, 60-90, and 110-150 kDa; three of these proteins were ZO-1, E-cadherin, and beta-catenin. Acetaldehyde reduced PTPase activity in plasma membrane and soluble fractions, whereas tyrosine kinase activity remained unaffected. Treatment with acetaldehyde resulted in a 97% loss of protein tyrosine phosphatase (PTP)1B activity and a partial reduction of PTP1C and PTP1D activities. These results strongly suggest that acetaldehyde inhibits PTPases to increase protein tyrosine phosphorylation, which may result in disruption of the tight junctions.     

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.     

Acetaldehyde dissociates the PTP1B-E-cadherin-beta-catenin complex in Caco-2 cell monolayers by a phosphorylation-dependent mechanism

Interactions between E-cadherin, beta-catenin and PTP1B (protein tyrosine phosphatase 1B) are crucial for the organization of AJs (adherens junctions) and epithelial cell-cell adhesion. In the present study, the effect of acetaldehyde on the AJs and on the interactions between E-cadherin, beta-catenin and PTP1B was determined in Caco-2 cell monolayers. Treatment of cell monolayers with acetaldehyde induced redistribution of E-cadherin and beta-catenin from the intercellular junctions by a tyrosine phosphorylation-dependent mechanism. The PTPase activity associated with E-cadherin and beta-catenin was significantly reduced and the interaction of PTP1B with E-cadherin and beta-catenin was attenuated by acetaldehyde. Acetaldehyde treatment resulted in phosphorylation of beta-catenin on tyrosine residues, and abolished the interaction of beta-catenin with E-cadherin by a tyrosine kinase-dependent mechanism. Protein binding studies showed that the treatment of cells with acetaldehyde reduced the binding of beta-catenin to the C-terminal region of E-cadherin. Pairwise binding studies using purified proteins indicated that the direct interaction between E-cadherin and beta-catenin was reduced by tyrosine phosphorylation of beta-catenin, but was unaffected by tyrosine phosphorylation of E-cadherin-C. Treatment of cells with acetaldehyde also reduced the binding of E-cadherin to GST (glutathione S-transferase)-PTP1B. The pairwise binding study showed that GST-E-cadherin-C binds to recombinant PTP1B, but this binding was significantly reduced by tyrosine phosphorylation of E-cadherin. Acetaldehyde increased the phosphorylation of beta-catenin on Tyr-331, Tyr-333, Tyr-654 and Tyr-670. These results show that acetaldehyde induces disruption of interactions between E-cadherin, beta-catenin and PTP1B by a phosphorylation-dependent mechanism.     

Independent regulation of adherens and tight junctions by tyrosine phosphorylation in Caco-2 cells

To study the role of tyrosine phosphorylation in the control of intercellular adhesion of intestinal cells, we have generated several clones of Caco-2 cells that express high levels of pp60v-src only after addition of butyrate. Expression of this oncogene in cells 5 days after confluence induced beta-catenin and p120-ctn tyrosine phosphorylation, redistribution of E-cadherin to the cytosol and disassembly of adherens junctions. However, tight junctions of Caco-2 cells at 5 days after confluence were not altered by expression of pp60v-src. Similar results were obtained when Caco-2 cells were incubated with phosphotyrosine phosphatase inhibitor orthovanadate. Although addition of this compound to postconfluent cells disrupt adherens junctions, tight junctions remain unaltered, as determined measuring monolayer permeability to mannitol or hyperphosphorylation of Triton-insoluble occludin. Modifications in tight junction permeability of Caco-2 were only observed at high concentrations of orthovanadate (1 mM). Interestingly, this tyrosine phosphorylation-refractory state was achieved after confluence since early postconfluent cells (day 2) showed a limited but significant response to low doses of orthovanadate. These results suggest that tight junctions of differentiated Caco-2 cells are uncoupled from adherens junctions and are insensitive to regulation by tyrosine phosphorylation.     

HGF regulates tight junctions in new nontumorigenic gastric epithelial cell line

The regulation of intercellular adhesion by hepatocyte growth factor (HGF) was examined on a novel nontumorigenic gastric epithelial cell line (IMGE-5) derived from H-2Kb-tsA58 transgenic mice. IMGE-5 cells constitutively expressed cytokeratin 18 and HGF receptors. Under permissive conditions (33 degrees C + interferon-gamma), IMGE-5 cells proliferated rapidly but did not display membrane expression of adherens and tight junction proteins. Under nonpermissive conditions, their proliferation was decreased and they displayed a strong, localized membrane expression of E-cadherin/beta-catenin and occludin/ZO-1. HGF treatment largely prevented the targeting of ZO-1 to the tight junction and induced a significant decrease of the transepithelial resistance measured across a confluent IMGE-5 cell monolayer. HGF rapidly increased the tyrosine phosphorylation of ZO-1 and decreased its association with occludin in a phosphatidylinositol 3-kinase (PI 3-kinase)-dependent manner. PI 3-kinase was also involved in HGF-induced migration of IMGE-5 cells. Our results demonstrate that 1) HGF prevents the appearance of ZO-1 in the membrane during epithelial cell differentiation; 2) HGF causes partial relocalization of ZO-1 to the cytoplasm and nucleus and concomitantly stimulates cell dissociation and migration; and 3) IMGE-5 cells offer a useful model for the study of gastric epithelial cell differentiation.     

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.     

PKC-δ binds to E-cadherin and mediates EGF-induced cell scattering

EGF is known to affect adherens junctions and disrupt cell-cell adhesion in a variety of carcinomas but the underlying mechanisms are not completely understood. Using human tumor epithelial cells overexpressing EGFR we demonstrated that EGF-induced cell scattering was mediated by protein kinase C-delta (PKC-δ). PKC-δ knockdown by siRNA significantly inhibited EGF-induced internalization of E-cadherin into the cytoplasm and blocked cell scattering. EGF phosphorylated PKC-δ at Y311 and ectopic expression of the mutant Y311F prevented PKC-δ binding to E-cadherin and EGF-induced cell scattering. Moreover, depletion of Src using siRNA decreased EGF-induced phosphorylation of PKC-δ at Y311 and blocked scattering. Finally, EGF reduced expression of the tight junction protein, occludin, and this effect was also mediated by PKC-δ through Src. In summary, PKC-δ mediated the effects of EGF on adherens and tight junctions thereby playing an important role in cell-cell adhesion with possible wider implications in tumor metastasis or epithelial-to-mesenchymal transition.     

Apical junction complex protein expression in the canine colon: differential expression of claudin-2 in the colonic mucosa in dogs with idiopathic colitis.

Canine idiopathic lymphocytic-plasmacytic colitis (LPC) is a well-recognized clinical and pathological entity in the dog, associated with altered immune cell populations and cytokine expression profiles. Clinical and experimental data indicate that alterations in the permeability of the intestinal epithelium contribute to the pathogenesis of a range of related conditions. The apical junction complex plays a significant role in regulating epithelial paracellular permeability, and we have characterized the distribution of a number of its component tight junction (ZO-1, occludin, claudin-2) and adherens junction (E-cadherin and beta-catenin) proteins in normal colon and colon from dogs with idiopathic LPC. ZO-1, occludin, E-cadherin, and beta-catenin exhibited a distribution in normal canine colon similar to that described previously in humans and rodents. In contrast to the situation in humans, claudin-2-specific labeling was observed in the normal canine colonic crypt epithelium, decreasing in intensity from the distal to the proximal crypt and becoming barely detectable at the luminal surface of the colon. There was little evidence for significant changes in ZO-1, occludin, E-cadherin, or beta-catenin expression in dogs affected by idiopathic LPC. However, claudin-2 expression markedly increased in the proximal crypt and luminal colonic epithelium in affected dogs, suggesting a role in the pathogenesis of canine LPC.     

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.     

Rho GTPase signaling regulates tight junction assembly and protects tight junctions during ATP depletion

Tight junctions control paracellular permeability and cellpolarity. Rho GTPase regulates tight junction assembly, and ATP depletion of Madin-Darby canine kidney (MDCK) cells (an in vitro modelof renal ischemia) disrupts tight junctions. The relationship between Rho GTPase signaling and ATP depletion was examined. Rho inhibition resulted in decreased localization of zonula occludens-1 (ZO-1) and occludin at cell junctions; conversely, constitutive Rhosignaling caused an accumulation of ZO-1 and occludin at cell junctions. Inhibiting Rho before ATP depletion resulted in more extensive loss of junctional components between transfected cells thancontrol junctions, whereas cells expressing activated Rho bettermaintained junctions during ATP depletion than control cells. ATPdepletion and Rho signaling altered phosphorylation signalingmechanisms. ZO-1 and occludin exhibited rapid decreases in phosphoaminoacid content following ATP depletion, which was restored on recovery.Expression of Rho mutant proteins in MDCK cells also altered levels ofoccludin serine/threonine phosphorylation, indicating that occludin isa target for Rho signaling. We conclude that Rho GTPase signalinginduces posttranslational effects on tight junction components. Ourdata also demonstrate that activating Rho signaling protects tightjunctions from damage during ATP depletion.

     

AMP-18 protects barrier function of colonic epithelial cells: role of tight junction proteins

Antrum mucosal protein (AMP)-18 and a synthetic peptide of amino acids 77-97 have mitogenic and motogenic properties for epithelial cells. The possibility that AMP-18 is also protective was evaluated in the colonic mucosa of mice and monolayer cultures of human colonic epithelial Caco-2/bbe (C2) cells. Administration of AMP peptide to mice with dextran sulfate sodium (DSS)-induced colonic injury delayed the onset of bloody diarrhea and reduced weight loss. Treatment of C2 cells with AMP peptide protected monolayers against decreases in transepithelial electrical resistance induced by the oxidant monochloramine, indomethacin, or DSS. A molecular mechanism for these barrier-protective effects was sought by asking whether AMP peptide acted on specific tight junction (TJ) proteins. Immunoblots of detergent-insoluble fractions of C2 cells treated with AMP peptide exhibited increased accumulation of specific TJ proteins. Occludin immunoreactivity was also increased in detergent-insoluble fractions obtained from colonic mucosal cells of mice injected with AMP peptide. Observations using laser scanning confocal (CF) microscopy supported the capacity of AMP peptide to enhance accumulation of occludin and zonula occludens-1 in TJ domains of C2 cell monolayers and together with immunoblot analysis showed that the peptide protected against loss of these TJ proteins following oxidant injury. AMP peptide also protected against a fall in TER during disruption of actin filaments by cytochalasin D and stabilized perijunctional actin during oxidant injury when assessed by CF. These findings suggest that AMP-18 could protect the intestinal mucosal barrier by acting on specific TJ proteins and stabilizing perijunctional actin.     

Glutamine supplementation attenuates ethanol-induced disruption of apical junctional complexes in colonic epithelium and ameliorates gut barrier dysfunction and fatty liver in mice

Previous in vitro studies showed that glutamine (Gln) prevents acetaldehyde-induced disruption of tight junctions and adherens junctions in Caco-2 cell monolayers and human colonic mucosa. In the present study, we evaluated the effect of Gln supplementation on ethanol-induced gut barrier dysfunction and liver injury in mice in vivo. Ethanol feeding caused a significant increase in inulin permeability in distal colon. Elevated permeability was associated with a redistribution of tight junction and adherens junction proteins and depletion of detergent-insoluble fractions of these proteins, suggesting that ethanol disrupts apical junctional complexes in colonic epithelium and increases paracellular permeability. Ethanol-induced increase in colonic mucosal permeability and disruption of junctional complexes were most severe in mice fed Gln-free diet. Gln supplementation attenuated ethanol-induced mucosal permeability and disruption of tight junctions and adherens junctions in a dose-dependent manner, indicating the potential role of Gln in nutritional intervention to alcoholic tissue injury. Gln supplementation dose-dependently elevated reduced-protein thiols in colon without affecting the level of oxidized-protein thiols. Ethanol feeding depleted reduced protein thiols and elevated oxidized protein thiols. Ethanol-induced protein thiol oxidation was most severe in mice fed with Gln-free diet and absent in mice fed with Gln-supplemented diet, suggesting that antioxidant effect is one of the likely mechanisms involved in Gln-mediated amelioration of ethanol-induced gut barrier dysfunction. Ethanol feeding elevated plasma transaminase and liver triglyceride, which was accompanied by histopathologic lesions in the liver; ethanol-induced liver damage was attenuated by Gln supplementation. These results indicate that Gln supplementation ameliorates alcohol-induced gut and liver injury.     

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.     

Differential regulation of junctional complex assembly in renal epithelial cell lines

Several signaling pathways that regulate tight junction and adherens junction assembly are being characterized. Calpeptin activates stress fiber assembly in fibroblasts by inhibiting SH2-containing phosphatase-2 (SHP-2), thereby activating Rho-GTPase signaling. Here, we have examined the effects of calpeptin on stress fiber and junctional complex assembly in Madin-Darby canine kidney (MDCK) and LLC-PK epithelial cells. Calpeptin induced disassembly of stress fibers and inhibition of Rho GTPase activity in MDCK cells. Interestingly, calpeptin augmented stress fiber formation in LLC-PK epithelial cells. Calpeptin treatment of MDCK cells resulted in a displacement of zonula occludens-1 (ZO-1) and occludin from cell-cell junctions and a loss of phosphotyrosine on ZO-1 and ZO-2, without any detectable effect on tight junction permeability. Surprisingly, calpeptin increased paracellular permeability in LLC-PK cells even though it did not affect tight junction assembly. Calpeptin also modulated adherens junction assembly in MDCK cells but not in LLC-PK cells. Calpeptin treatment of MDCK cells induced redistribution of E-cadherin and -catenin from intercellular junctions and reduced the association of p120ctn with the E-cadherin/catenin complex. Together, our studies demonstrate that calpeptin differentially regulates stress fiber and junctional complex assembly in MDCK and LLC-PK epithelial cells, indicating that these pathways may be regulated in a cell line-specific manner. calpeptin; tight junctions; adherens junctions; Rho; cadherin; p120ctn     

Glucocorticoid down-regulation of RhoA is required for the steroid-induced organization of the junctional complex and tight junction formation in rat mammary epithelial tumor cells

In Con8 mammary epithelial tumor cells, we have documented previously that the synthetic glucocorticoid dexamethasone induces the reorganization of the tight junction and adherens junction (apical junction) and stimulates the monolayer transepithelial electrical resistance (TER), which is a reliable in vitro measurement of tight junction sealing. Western blots demonstrated that dexamethasone treatment down-regulated the level of the RhoA small GTPase prior to the stimulation of the monolayer TER. To test the role of RhoA in the steroid regulation of apical junction dynamics functionally, RhoA levels were altered in Con8 cells by transfection of either constitutively active (RhoA.V14) or dominant negative (RhoA.DN19) forms of RhoA. Ectopic expression of constitutively active RhoA disrupted the dexamethasone-stimulated localization of zonula occludens-1 and beta-catenin to sites of cell-cell contact, inhibited tight junction sealing, and prevented the complete formation of the F-actin ring structure at the apical side of the cell monolayer. In a complementary manner, dominant negative RhoA caused a precocious organization of the tight junction, adherens junction, and the F-actin rings in the absence of steroid, whereas the monolayer TER remained glucocorticoid-responsive. Taken together, our results demonstrate that the glucocorticoid down-regulation of RhoA is a required step in the steroid signaling pathway which controls the organization of the apical junctional complex and the actin cytoskeleton in mammary epithelial cells.     

Differentiation of the epithelial apical junctional complex during mouse preimplantation development: a role for rab13 in the early maturation of the tight junction

We have investigated the mechanisms by which the epithelial apicolateral junctional complex (AJC) is generated during trophectoderm differentiation in the mouse blastocyst using molecular, structural and functional analyses. The mature AJC comprises an apical tight junction (TJ), responsible for intercellular sealing and blastocoel formation, and subjacent zonula adherens E-cadherin/catenin adhesion complex which also extends along lateral membrane contact sites. Dual labelling confocal microscopy revealed that the AJC derived from a single 'intermediate' complex formed following embryo compaction at the 8-cell stage in which the TJ-associated peripheral membrane protein, ZO-1alpha- isoform, was co-localized with both alpha- and beta-catenin. However, following assembly of the TJ transmembrane protein, occludin, from the early 32-cell stage when blastocoel formation begins, ZO-1alpha- and other TJ proteins (ZO-1alpha+ isoform, occludin, cingulin) co-localized in an apical TJ which was separate from a subjacent E-cadherin/catenin zonula adherens complex. Thin-section electron microscopy confirmed that a single zonula adherens-like junctional complex present at the AJC site following compaction matured into a dual TJ and zonula adherens complex at the blastocyst stage. Embryo incubation in the tracer FITC-dextran 4 kDa showed that a functional TJ seal was established coincident with blastocoel formation. We also found that rab13, a small GTPase previously localized to the TJ, is expressed at all stages of preimplantation development and relocates from the cytoplasm to the site of AJC biogenesis from compaction onwards with rab13 and ZO-1alpha- co-localizing precisely. Our data indicate that the segregation of the two elements of the AJC occurs late in trophectoderm differentiation and likely has functional importance in blastocyst formation. Moreover, we propose a role for rab13 in the specification of the AJC site and the formation and segregation of the TJ.     

The protein tyrosine phosphatase Pez is a major phosphatase of adherens junctions and dephosphorylates beta-catenin

Cell-cell adhesion regulates processes important in embryonal development, normal physiology, and cancer progression. It is regulated by various mechanisms including tyrosine phosphorylation. We have previously shown that the protein tyrosine phosphatase Pez is concentrated at intercellular junctions in confluent, quiescent monolayers but is nuclear in cells lacking cell-cell contacts. We show here with an epithelial cell model that Pez localizes to the adherens junctions in confluent monolayers. A truncation mutant lacking the catalytic domain acts as a dominant negative mutant to upregulate tyrosine phosphorylation at adherens junctions. We identified beta-catenin, a component of adherens junctions, as a substrate of Pez by a "substrate trapping" approach and by in vitro dephosphorylation with recombinant Pez. Consistent with this, ectopic expression of the dominant negative mutant caused an increase in tyrosine phosphorylation of beta-catenin, demonstrating that Pez regulates the level of tyrosine phosphorylation of adherens junction proteins, including beta-catenin. Increased tyrosine phosphorylation of adherens junction proteins has been shown to decrease cell-cell adhesion, promoting cell migration as a result. Accordingly, the dominant negative Pez mutant enhanced cell motility in an in vitro "wound" assay. This suggests that Pez is also a regulator of cell motility, most likely through its action on cell-cell adhesion.     

Oxidized phospholipids mediate occludin expression and phosphorylation in vascular endothelial cells

Oxidized l-alpha-1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (OxPAPC), a component of minimally modified LDL, induces production of proinflammatory cytokines and development of atherosclerotic lesions. We tested the hypothesis that OxPAPC alters expression, phosphorylation, and localization of tight junction (TJ) proteins, particularly occludin, a transmembrane TJ protein. OxPAPC reduced total occludin protein and increased occludin phosphorylation dose dependently (10-50 microg/ml) and time dependently in bovine aortic endothelial cells. OxPAPC decreased occludin mRNA and reduced the immunoreactivity of zonula occludens-1 at the cell-cell contacts. Furthermore, OxPAPC increased the diffusive flux of 10-kDa dextran in a dose-dependent manner. O2-* production by bovine aortic endothelial cells increased nearly twofold after exposure to OxPAPC. Also, enzymatic generation of O2-* by xanthine oxidase-lumazine and H2O2 by glucose oxidase-glucose increased occludin phosphorylation, implicating reactive oxygen species as modulators of the OxPAPC effects on occludin phosphorylation. Superoxide dismutase and/or catalase blocked the effects of OxPAPC on occludin protein content and phosphorylation, occludin mRNA, zonula occludens-1 immunoreactivity, and diffusive flux of 10-kDa dextran. These findings suggest that changes in TJ proteins are potential mechanisms by which OxPAPC compromises the barrier properties of the vascular endothelium. OxPAPC-induced disruption of TJs, which likely facilitates transmigration of LDL and inflammatory cells into the subendothelial layers, may be mediated by reactive oxygen species.