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At least 703 records · Page 39Linked to original sources
PubMed1991

Rapid aggregation and tight junction formation in single cell suspensions of tumor cells after very low dose trypsin treatment.

The possible participation of tight junction formation in intercellular adhesion of tumor cells was studied in single cell suspensions of HT29 adenocarcinoma cells. Very low dose trypsin treatment (0.15 micrograms/ml, 30 min, 37 degrees C) induced rapid intercellular adhesion of suspended HT29 colon carcinoma cells. Intercellular adhesion was independent of the presence of divalent cations. Electron microscopy of freeze-fractured membrane fragments of trypsin-treated HT29 cells revealed a dramatic increase of typical tight junction structures during aggregation.

Adenocarcinoma↗
PubMed1992

The impenetrability of 5-(N-hexadecanoyl)aminofluoroscein through endothelial cell monolayers is dependent upon its solution properties, not the presence of tight junctions.

The solution properties of two fluorescent lipophilic analogues were examined in conjunction with their ability to penetrate the tight junctions of bovine aortic endothelial cell monolayers. 5-(N-dodecanoyl)aminofluoroscein was shown to label both the apical and basolateral plasma membrane domains of confluent monolayers at 4 degrees C and pH 7.3, but 5-(N-hexadecanoyl)aminofluoroscein was shown to label only the apical membrane domain. When used under more soluble conditions at 20 degrees C and pH 8.5, both probes labeled apical and basolateral plasma membrane domains more equally. This indicates that solubility conditions, and not tight junctions, dictate the penetration of 5-(N-hexadecanoyl)aminofluoroscein from the apical to the basolateral plasma membrane domain.

Animals↗
PubMed2004

Rab13 regulates PKA signaling during tight junction assembly.

The GTPase Rab13 regulates the assembly of functional epithelial tight junctions (TJs) through a yet unknown mechanism. Here, we show that expression of the GTP-bound form of Rab13 inhibits PKA-dependent phosphorylation and TJ recruitment of the vasodilator-stimulated phosphoprotein, an actin remodelling protein. We demonstrate that Rab13GTP directly binds to PKA and inhibits its activity. Interestingly, activation of PKA abrogates the inhibitory effect of Rab13 on the recruitment of vasodilator-stimulated phosphoprotein, ZO-1, and claudin1 to cell-cell junctions. Rab13 is, therefore, the first GTPase that controls PKA activity and provides an unexpected link between PKA signaling and the dynamics of TJ assembly.

Animals↗
PubMed1998

Effects of beta-lactoglobulin on the tight-junctional stability of Caco-2-SF monolayer.

The mechanisms for tight-junction (TJ) stabilization by beta-lactoglobulin (beta-Lg) were studied. Treatment of Caco-2-SF cells with inhibitors for some enzymes in the intracellular signal transduction pathways and a cytoskeleton-disturbing agent (cytochalasin D) reduced the TJ-stabilizing activity of beta-Lg. So beta-Lg is suggested to modulate the cytoskeletal structure through the activation of phospholipase C and protein kinase C, resulting in the TJ stabilization.

Animals↗
PubMed2006

Tight junction proteins in the skin.

It has long been accepted that tight junctions (TJ) are crucial for the formation and maintenance of the paracellular barrier and for cell polarity in simple epithelia and endothelia. Moreover, it is long known that they play a role in barrier function of amphibian skin. However, only in recent years were TJ and TJ proteins identified in the epidermis of men and mice. Their involvement in the barrier function of mammalian skin has been shown. This review summarizes our current knowledge about TJ and TJ proteins in mammalian skin.

Animals↗
PubMed2003

The organization of tight junctions in epithelia: implications for mammary gland biology and breast tumorigenesis.

Tight junctions (TJs), the most apical components of the cell-cell junctional complexes, play a crucial role in the establishment and maintenance of cell polarity within tissues. In secretory glandular tissues, such as the mammary gland, TJs are crucial for separating apical and basolateral domains. TJs also create the variable barrier regulating paracellular movement of molecules through epithelial sheets, thereby maintaining tissue homeostasis. Recent advances reveal that TJs exist as macromolecular complexes comprised of several types of membrane proteins, cytoskeletal proteins, and signaling molecules. Many of these components are regulated during mammary gland development and pregnancy cycles, and several have received much attention as possible "tumor suppressors" during progression to breast cancer.

Animals↗
PubMed2006

The heat-shock protein Apg-2 binds to the tight junction protein ZO-1 and regulates transcriptional activity of ZONAB.

The tight junction adaptor protein ZO-1 regulates intracellular signaling and cell proliferation. Its Src homology 3 (SH3) domain is required for the regulation of proliferation and binds to the Y-box transcription factor ZO-1-associated nucleic acid binding protein (ZONAB). Binding of ZO-1 to ZONAB results in cytoplasmic sequestration and hence inhibition of ZONAB's transcriptional activity. Here, we identify a new binding partner of the SH3 domain that modulates ZO-1-ZONAB signaling. Expression screening of a cDNA library with a fusion protein containing the SH3 domain yielded a cDNA coding for Apg-2, a member of the heat-shock protein 110 (Hsp 110) subfamily of Hsp70 heat-shock proteins, which is overexpressed in carcinomas. Regulated depletion of Apg-2 in Madin-Darby canine kidney cells inhibits G(1)/S phase progression. Apg-2 coimmunoprecipitates with ZO-1 and partially localizes to intercellular junctions. Junctional recruitment and coimmunoprecipitation with ZO-1 are stimulated by heat shock. Apg-2 competes with ZONAB for binding to the SH3 domain in vitro and regulates ZONAB's transcriptional activity in reporter gene assays. Our data hence support a model in which Apg-2 regulates ZONAB function by competing for binding to the SH3 domain of ZO-1 and suggest that Apg-2 functions as a regulator of ZO-1-ZONAB signaling in epithelial cells in response to cellular stress.

Animals↗
PubMed2004

Expression of occludin, a tight-junction-associated protein, in human lung carcinomas.

Occludin is a tight-junction-associated transmembrane protein, and previous observations suggested that occludin might play a crucial role in the formation and maintenance of organized tubular structures. Based on these observations, we explored the possible role of occludin immunostaining in the diagnosis of lung carcinomas. A total of 68 lung carcinomas and surrounding normal lung tissues were studied. A formalin-fixed, paraffin-embedded section from each tumor was stained with a new anti-occludin monoclonal antibody raised in our laboratory. In normal lung tissues, the anti-occludin antibody strongly stained the apicoluminal borders of the bronchial/bronchiolar epithelia and bronchial glands as a dot or short line. The antibody also stained the intercellular borders of alveolar epithelia. In cancer cells that faced lumina of all adenocarcinomas, regardless of grade, including bronchioloalveolar carcinomas, occludin showed an expression pattern identical to that of the normal bronchial and alveolar epithelia. Occludin reactivity was not noted in any cases of squamous cell carcinoma, large cell carcinoma, small cell carcinoma, or large cell neuroendocrine carcinoma. The results of the present study suggest that occludin can serve as an immunohistochemical indicator of the "true" glandular differentiation that forms tubulo-papillary structures in human lung carcinoma tissues.

Humans↗
PubMed2000

Disruption of epithelial tight junctions is prevented by cyclic nucleotide-dependent protein kinase inhibitors.

Tight junctions (TJs), the most apical of the intercellular junctions, prevent the passage of ions and molecules through the paracellular pathway. Intracellular signalling molecules are likely to be involved in the regulation of TJ integrity. In order to specifically investigate the role of protein kinase A (PKA) in the maintenance of epithelial TJ integrity, calcium-switch experiments were performed, in which calcium was removed from EpH4 and MDCK culture medium, in the absence or presence of the PKA inhibitors H-89 or HA-1004. Removal of calcium from the culture media of the epithelial cells resulted in disruption of the TJs, characterised by a loss of membrane association of the TJ-associated proteins occludin, ZO-1 and ZO-2, by a loss of TJ strands, by a marked decrease in the transepithelial electrical resistance and by a dramatic increase in the transepithelial permeability to tracers. The association of occludin, ZO-1 and ZO-2 with the actin cytoskeleton is not affected. In contrast, when the removal of calcium was performed in the presence of either the PKA inhibitor H-89 or HA-1004, all barrier characteristics were preserved. Our data indicate that following the removal of calcium from the culture medium of epithelial cells in vitro, PKA is activated and subsequently is involved in the disruption of TJs.

Actins↗
PubMed2006

Therapeutic utility of a novel tight junction modulating peptide for enhancing intranasal drug delivery.

Previously, a novel tight junction modulating (TJM) peptide was described affording a transient, reversible lowering of transepithelial electrical resistance (TER) in an in vitro model of nasal epithelial tissue. In the current report, this peptide has been further evaluated for utility as an excipient in transepithelial drug formulations. Chemical stability was optimal at neutral to acidic pH when stored at or below room temperature, conditions relevant to therapeutic formulations. The TJM peptide was tested in the in vitro tissue model for potential to enhance permeation of a low-molecular-weight (LMW) drug, namely the acetylcholinesterase inhibitor galantamine, as well as three peptides, salmon calcitonin, parathyroid hormone 1-34 (PTH(1-34)), and peptide YY 3-36 (PYY(3-36)). In all cases, the TJM peptide afforded a dramatic improvement in drug permeation across epithelial tissue. In addition, a formulation containing PYY(3-36) and TJM peptide was dosed intranasally in rabbits, resulting in a dramatic increase in bioavailability. The TJM peptide was as or more effective in enhancing PYY(3-36) permeation in vivo at a 1000-fold lower molar concentration compared to using LMW enhancers. Based on these in vitro and in vivo data, the novel TJM peptide represents a promising advancement in intranasal formulation development.

Administration, Intranasal↗
PubMed2006

The tight junction protein ZO-2 has several functional nuclear export signals.

The tight junction (TJ) protein ZO-2 changes its subcellular distribution according to the state of confluency of the culture. Thus in confluent monolayers, it localizes at the TJ region whereas in sparse cultures it concentrates at the nucleus. The canine sequence of ZO-2 displays four putative nuclear export signals (NES), two at the second PDZ domain (NES-0 and NES-1) and the rest at the GK region (NES-2 and NES-3). The functionality of NES-0 and NES-3 was unknown, hence here we have explored it with a nuclear export assay, injecting into the nucleus of MDCK cells peptides corresponding to the ZO-2 NES sequences chemically coupled to ovalbumin. We show that both NES-0 and NES-3 are functional and sensitive to leptomycin B. We also demonstrate that NES-1, previously characterized as a non functional NES, is rendered capable of nuclear export upon the acquisition of a negative charge at its Ser369 residue. Experiments performed injecting at the nucleus WT and mutated ZO-2-GST fusion proteins revealed the need of both NES-0 and NES-1, and NES-2 and NES-3 for attaining an efficient nuclear exit of the respective amino and middle segments of ZO-2. Moreover, the transfection of MDCK cells with full-length ZO-2 revealed that the mutation of any of the NES present in the molecule was sufficient to induce nuclear accumulation of the protein.

Active Transport, Cell Nucleus↗
PubMed2003

Matrix metalloproteinase-9-deficient dendritic cells have impaired migration through tracheal epithelial tight junctions.

When sampling inhaled antigens, dendritic cells (DC) must penetrate the tight junction (TJ) barrier while maintaining the TJ seal. In matrix metalloproteinase (MMP)-9-deficient mice, in vivo experiments suggest that migration of DC into air spaces is impaired. To examine the underlying mechanisms, we established a well-defined in vitro model using mouse tracheal epithelial cells and mouse bone marrow DC (BMDC). Transmigration was elicited with either macrophage inflammatory protein (MIP)-1alpha or MIP-3beta in a time-dependent manner. Control MMP-9(+/+) BMDC cultured with granulocyte macrophage-colony-stimulating factor for 7 d showed a 30-fold greater transepithelial migration toward MIP-3beta than MIP-1alpha, indicating a more mature DC phenotype. MMP-9(-/-) BMDC as well as MMP-9(+/+) BMDC in the presence of the MMP inhibitor GM6001, although showing a similar preference for MIP-3beta, were markedly impaired in their ability to traverse the epithelium. Expression levels of CCR5 and CCR7, however, were similar in both MMP-9(-/-) and MMP-9(+/+) BMDC. Expression of the integral TJ proteins, occludin and claudin-1, were examined in BMDC before and after transepithelial migration. Interestingly, occludin but not claudin-1 was degraded following transepithelial migration in both MMP-9(-/-) and control BMDC. In addition, there was a > 2-fold increase in claudin-1 expression in MMP-9(-/-) as compared with control BMDC. These observations indicate that occludin and claudin-1 are differentially regulated and suggest that the lack of MMP-9 may affect claudin-1 turnover.

Animals↗
PubMed2002

Occludin TM4(-): an isoform of the tight junction protein present in primates lacking the fourth transmembrane domain.

The tight junction protein occludin possesses four transmembrane domains, two extracellular loops, and cytoplasmic N- and C-termini. Reverse transcription-PCR analysis of human tissues, embryos and cells using primers spanning the fourth transmembrane domain (TM4) and adjacent C-terminal region revealed two products. The larger and predominant product corresponded in sequence to canonical occludin (TM4(+)), while the smaller product exhibited a 162 bp deletion encoding the entire TM4 and immediate C-terminal flanking region (TM4(-)). Examination of the genomic occludin sequence identified that the 162 bp sequence deleted in TM4(-) coincided precisely with occludin exon 4, strongly suggesting that TM4(-) is an alternative splice isoform generated by skipping of exon 4. Indeed, the reading frame of downstream exons is not affected by exclusion of exon 4. The presence of both TM4(+) and TM4(-) occludin isoforms was also identified in monkey epithelial cells but TM4(-) was undetected in murine and canine tissue and cells, indicating a late evolutionary origin for this alternative splicing event. Conceptual translation of TM4(-) isoform predicts extracellular localisation of the C-terminus. Immunocytochemical processing of living human Caco-2 cells using a C-terminal occludin antibody revealed weak, discontinuous staining restricted to the periphery of subconfluent islands of cells, or islands generated by wounding confluent layers. In occludin immunoblots, a weak band at approximately 58 kDa, smaller than the predominant band at 65 kDa and corresponding to the predicted mass of TM4(-) isoform, is evident and upregulated in subconfluent cells. These data suggest that the TM4(-) isoform may be translated at low levels in specific conditions and may contribute to regulation of occludin function.

Alternative Splicing↗
PubMed1999

A role for intracellular calcium in tight junction reassembly after ATP depletion-repletion.

The integrity of the tight junction (TJ), which is responsible for the permeability barrier of the polarized epithelium, is disrupted during ischemic injury and must be reestablished for recovery. Recently, with the use of an ATP depletion-repletion model for ischemia and reperfusion injury in Madin-Darby canine kidney cells, TJ proteins such as zonula occludens-1 (ZO-1) were shown to reversibly form large complexes and associate with cytoskeletal proteins (T. Tsukamoto and S. K. Nigam, J. Biol. Chem. 272: 16133-16139, 1997). In this study, we examined the role of intracellular calcium in TJ reassembly after ATP depletion-repletion by employing the cell-permeant calcium chelator 1, 2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-AM (BAPTA-AM). Lowering intracellular calcium during ATP depletion is associated with significant inhibition of the reestablishment of the permeability barrier following ATP repletion as measured by transepithelial electrical resistance and mannitol flux, marked alterations in the subcellular localization of occludin by immunofluorescent analysis, and decreased solubility of ZO-1 and other TJ proteins by Triton X-100 extraction assay, suggesting that lowering intracellular calcium potentiates the interaction of TJ proteins with the cytoskeleton. Coimmunoprecipitation studies indicated that decreased solubility may partly result from the stabilization of large TJ protein-containing complexes with fodrin. Although ionic detergents (SDS and deoxycholate) appeared to cause a dissociation of ZO-1-containing complexes from the cytoskeleton, sucrose gradient analyses of the solubilized proteins suggested that calcium chelation leads to self-association of these complexes. Together, these results raise the possibility that intracellular calcium plays an important facilitatory role in the reassembly of the TJ damaged by ischemic insults. Calcium appears to be necessary for the dissociation of TJ-cytoskeletal complexes, thus permitting functional TJ reassembly and paracellular permeability barrier recovery.

Adenosine Triphosphate↗
PubMed2006

Molecular mechanism of tumor necrosis factor-alpha modulation of intestinal epithelial tight junction barrier.

A TNF-alpha-induced increase in intestinal epithelial tight junction (TJ) permeability has been proposed to be an important proinflammatory mechanism contributing to intestinal inflammation in Crohn's disease and other inflammatory conditions. Previous studies from our laboratory suggested that the TNF-alpha-induced increase in intestinal TJ permeability was mediated by an increase in myosin light chain kinase (MLCK) protein expression. However, the molecular mechanisms that mediate the TNF-alpha increase in intestinal TJ permeability and MLCK protein expression remain unknown. The purpose of this study was to delineate the intracellular and molecular mechanisms that mediate the TNF-alpha-induced increase in intestinal TJ permeability; using an in vitro intestinal epithelial model system consisting of filter-grown Caco-2 intestinal epithelial monolayers. To examine the molecular mechanisms involved in the TNF-alpha regulation of intestinal TJ barrier, we identified and cloned for the first time a functionally active MLCK promoter region. TNF-alpha treatment of filter-grown Caco-2 monolayers transfected with plasmid vector containing the MLCK promoter region produced an increase in MLCK promoter activity and MLCK transcription. The TNF-alpha-induced increase in MLCK transcription corresponded to a sequential increase in MLCK protein expression, MLCK activity, and Caco-2 TJ permeability. The TNF-alpha-induced increase in MLCK promoter activity was mediated by NF-kappaB activation, and the inhibition of NF-kappaB activation prevented the TNF-alpha-induced increase in promoter activity and the subsequent increase in MLCK protein expression and Caco-2 TJ permeability. The TNF-alpha-induced activation of MLCK promoter was mediated by binding of the activated NF-kappaB p50/p65 dimer to the downstream kappaB binding site (-84 to -75) on the MLCK promoter region; deletion of the kappaB binding site prevented the TNF-alpha increase in promoter activity. Additionally, siRNA silencing of NF-kappaB p65 also prevented the TNF-alpha increase in MLCK promoter activity. In conclusion, our findings indicated that the TNF-alpha-induced increase in intestinal epithelial TJ permeability was mediated by NF-kappaB p50/p65 binding and activation of the MLCK promoter. NF-kappaB p50/p65 activation of the MLCK promoter then leads to a stepwise increase in MLCK transcription, expression and activity, and MLCK-mediated opening of the intestinal TJ barrier.

Caco-2 Cells↗
PubMed2006

Par-3 mediates the inhibition of LIM kinase 2 to regulate cofilin phosphorylation and tight junction assembly.

The polarity protein Par-3 plays critical roles in axon specification and the establishment of epithelial apico-basal polarity. Par-3 associates with Par-6 and atypical protein kinase C and is required for the proper assembly of tight junctions, but the molecular basis for its functions is poorly understood. We now report that depletion of Par-3 elevates the phosphorylated pool of cofilin, a key regulator of actin dynamics. Expression of a nonphosphorylatable mutant of cofilin partially rescues tight junction assembly in cells lacking Par-3, as does the depletion of LIM kinase 2 (LIMK2), an upstream kinase for cofilin. Par-3 binds to LIMK2 but not to the related kinase LIMK1. Par-3 inhibits LIMK2 activity in vitro, and overexpressed Par-3 suppresses cofilin phosphorylation that is induced by lysophosphatidic acid. Our findings identify LIMK2 as a novel target of Par-3 and uncover a molecular mechanism by which Par-3 could regulate actin dynamics during cell polarization.

Actin Depolymerizing Factors↗
PubMed1992

Epithelial permeability factor: a serum protein that condenses actin and opens tight junctions.

An epithelial permeability factor (EPF) in human serum lowered, within 1 h, the transepithelial electrical resistance and opened the tight junctions of a cultured kidney epithelium (Madin-Darby canine kidney) when it came in contact with the basolateral surface of the kidney epithelium. Size-exclusion chromatography of serum or heat-inactivated serum resolved seven peaks of EPF activity (approximately 15, approximately 30, approximately 45, approximately 60, approximately 120, and approximately 240 kDa and greater than 240 kDa) with 65% of the activity at approximately 45, approximately 60, and approximately 120 kDa. Heat inactivation, which had no effect on total activity, caused a significant decrease in the activity at 120 kDa and an equivalent rise in activity at 45 kDa. Although acid charcoal extraction or lectin affinity chromatography did not remove activity, EPF activity was eliminated by pepsin. Heat-inactivated serum or fractions containing EPF had no effect on ZO-1 localization but did cause a dose-dependent focal condensation of the perijunctional actin ring at sites where three or more cells were in contact. These data suggest that EPF is a protein that appears to form multimers that interact with the basolateral surface of the epithelium and cause constriction of the cytoskeleton and an increase in permeability at specific sites along the tight junction.

Actins↗
PubMed1989

Effect of tight junctional resistance on penetration of LLC-PK1 epithelial cell layers by metastatic B16-F10 melanoma cells.

The relationship between tight junctional resistance of a tissue and its penetration by metastatic cells was examined in vitro using LLC-PK1 cells, an epithelial cell line derived from pig kidney, and B16-F10 cells, a murine melanoma cell line metastatic in syngeneic C57BL/6 mice. When grown to confluence on 8.0-microns pore size polycarbonate filters, LLC-PK1 cells formed tight junctions between adjacent cells which offered an electrical resistance to a nondestructive 20-mu ampere alternating current passed across the cell layer. B16-F10 cells seeded on top of an LLC-PK1 epithelial layer with a measured transepithelial resistance of approximately 300 omega x cm2 were unable to penetrate the epithelial barrier. The tumor-promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) lowered the electrical resistance of the epithelial layer by 80% and simultaneously opened the epithelial barrier to penetration by metastatic cells.

Animals↗