Disruption Of Intercellular Junctions Research Articles

Induction and regulation of epithelial–mesenchymal transitions

Herein we discuss the factors that bring about the transformation of epithelial cells into cells of fibroblastic phenotype. This type of transformation, referred to as epithelium-to-mesenchyme transition (EMT), allows cells to dissociate from the epithelial tissue from which they originate and to migrate freely. EMT is therefore thought to play a fundamental role during the early steps of invasion and metastasis of carcinoma cells. Among biological agents which have been identified as inducers of EMT are a number of cytokines and extracellular matrix macromolecules. The coordinated changes in cell morphology, associated with the induction of cell motility and the disruption of intercellular junctions, are the consequence of a signaling cascade emanating from the plasma membrane and leading to changes in gene expression. Understanding the mechanisms regulating EMT of normal and transformed epithelial cells may offer new perspectives for designing therapies for the treatment of metastatic cancers of epithelial origin.

Human junction adhesion molecule regulates tight junction resealing in epithelia.

Epithelial cells form a highly selective barrier and line many organs. The epithelial barrier is maintained by closely apposed cell-cell contacts containing tight junctions, the regulation of which is incompletely understood. Here we report the cloning, tissue localization and evidence for a role in epithelial barrier regulation of an immunoglobulin superfamily member that likely represents the human homolog of murine junction adhesion molecule (JAM). Analysis of the primary structure of human JAM, cloned from T84 epithelial cells, predicts a transmembrane protein with an extracellular domain that contains two IgV loops. Monoclonal antibodies generated against the putative extracellular domain were reactive with a 35-39 kDa protein from both T84 epithelial cells and human neutrophils. By immunofluorescence, JAM mAbs labeled epithelial cells from intestine, lung, and kidney, prominently in the region of tight junctions (co-localization with occludin) and also along lateral cell membranes below the tight junctions. Flow cytometric studies confirmed predominant JAM expression in epithelial cells but also revealed expression on endothelial and hematopoietic cells of all lineages. Functional studies demonstrated that JAM specific mAbs markedly inhibited transepithelial resistance recovery of T84 monolayers after disruption of intercellular junctions (including tight junctions) by transient calcium depletion. Morphologic analysis revealed that, after disassembly of cell-cell junctions, anti-JAM inhibition of barrier function recovery correlated with a loss of both occludin and JAM, but not ZO-1, in reassembling tight junction structure. Reassembly of the major adherens junction component E-cadherin was not affected by JAM specific mAbs. Our findings suggest that JAM plays an important role in the regulation of tight junction assembly in epithelia. Furthermore, these JAM-mediated effects may occur by either direct, or indirect interactions with occludin.

Selective degradation of E-cadherin and dissolution of E-cadherin-catenin complexes in epithelial ischemia.

Ischemic epithelial cells are characterized by disruption of intercellular junctions and loss of apical-basolateral protein polarity, which are normally dependent on the integrity of the adherens junction (AJ). Biochemical analysis of both whole ischemic kidneys and ATP-depleted Madin-Darby canine kidney (MDCK) cells demonstrated a striking loss of E-cadherin (the transmembrane protein of the AJ) with the appearance and accumulation of an approximately 80-kDa fragment reactive with anti-E-cadherin antibodies on Western blots of ATP-depleted MDCK cells. This apparent ischemia-induced degradation of E-cadherin was not blocked by either inhibitors of the major proteolytic pathways (i.e., proteasome, lysosome, or calpain), or by chelation of intracellular calcium, suggesting the involvement of a protease capable of functioning at low ATP and low calcium levels. Immunocytochemistry revealed the movement of several proteins normally comprising the AJ, including E-cadherin and beta-catenin, away from lateral portions of the plasma membrane to intracellular sites. Moreover, rate-zonal centrifugation and immunoprecipitation with anti-E-cadherin and anti-beta-catenin antibodies indicated that ATP depletion disrupted normal E-cadherin-catenin interactions, resulting in the dissociation of alpha- and gamma-catenin from E-cadherin and beta-catenin-containing complexes. Because the generation and maintenance of polarized epithelial cells are dependent upon E-cadherin-mediated cell-cell adhesion and normal AJ function, we propose that the rapid degradation of E-cadherin and dissolution of the AJ is a key step in the development of the ischemic epithelial cell phenotype. Furthermore, we hypothesize that the reassembly of the AJ after ischemia/ATP depletion may require a novel bioassembly mechanism involving recombination of newly synthesized and sorted E-cadherin with preexisting pools of catenins that have (temporally) redistributed intracellularly.

Trefoil family factor 2(TFF2) results in disruption of intercellular junctions facilitating cell migration and restitution

Trefoil family factor 2(TFF2) results in disruption of intercellular junctions facilitating cell migration and restitution

Molecular cloning and characterization of prostase, an androgen-regulated serine protease with prostate-restricted expression

The identification of genes with selective expression in specific organs or cell types provides an entry point for understanding biological processes that occur uniquely within a particular tissue. Using a subtraction approach designed to identify genes preferentially expressed in specific tissues, we have identified prostase, a human serine protease with prostate-restricted expression. The prostase cDNA encodes a putative 254-aa polypeptide with a conserved serine protease catalytic triad and an amino-terminal pre-propeptide sequence, indicating a potential secretory function. The genomic sequence comprises five exons and four introns and contains multiple copies of a chromosome 19q-specific minisatellite repeat. Northern analysis indicates that prostase mRNA is expressed in hormonally responsive normal and neoplastic prostate epithelial tissues, but not in prostate stromal constituents. Prostase shares 35% amino acid identity with prostate-specific antigen (PSA) and 78% identity with the porcine enamel matrix serine proteinase 1, an enzyme involved in enamel matrix degradation and with a putative role in the disruption of intercellular junctions. Radiation-hybrid-panel mapping localized prostase to chromosome 19q13, a region containing several other serine proteases, including protease M, pancreatic/renal kallikrein hK1, and the prostate-specific kallikreins hK2 and hK3 (PSA). The sequence homology between prostase and other well-characterized serine proteases suggests several potential functional roles for the prostase protein that include the degradation of extracellular matrix and the activation of PSA and other proteases.

Dephosphorylation of the Cadherin-associated p100/p120 Proteins in Response to Activation of Protein Kinase C in Epithelial Cells

Protein kinase C signaling pathways have been implicated in the disruption of intercellular junctions, but mechanisms are not clear. p100 and p120 are members of the Armadillo family of proteins and are localized to cellular adherens junctions. In strain I Madin-Darby canine kidney cells, protein kinase C activation leads to disruption of tight junctions and an increase in permeability of cell monolayers. We show that this permeability increase is accompanied by dephosphorylation of p100/p120 on serine and threonine residues. The dephosphorylation of these proteins can also be induced by the kinase inhibitors staurosporine, KT5926, and Gö 6976. Treatment of cells with phosphatase inhibitors induced hyperphosphorylation of p100 and p120. Thus, p100 and p120 participate in a regulatable cycle of serine/threonine phosphorylation and dephosphorylation. Protein kinase C must act, directly or indirectly, by perturbing this phosphorylation cycle, by inhibition of a p100/p120 kinase and/or activation of a phosphatase. These data clearly show that p100 and p120 are targets of a novel protein kinase C signaling pathway. Dephosphorylation of these proteins precedes the permeability increase across epithelial cell monolayers seen in response to phorbol esters, raising the possibility that this pathway may play a role in the modulation of intercellular junctions.

The estrogen-dependent c-JunER protein causes a reversible loss of mammary epithelial cell polarity involving a destabilization of adherens junctions.

Members of the epidermal growth factor (EGF) receptor family are known to be specifically involved in mammary carcinogenesis. As a nuclear target of activated receptors, we examined c-Jun in mammary epithelial cells. For this, we used a c-JunER fusion protein which was tightly controlled by estrogen. Activation of the JunER by hormone resulted in the transcriptional regulation of a variety of AP-1 target genes. Hormone-activated JunER induced the loss of epithelial polarity, a disruption of intercellular junctions and normal barrier function and the formation of irregular multilayers. These changes were completely reversible upon hormone withdrawal. Loss of epithelial polarity involved redistribution of both apical and basolateral proteins to the entire plasma membrane. The redistribution of E-cadherin and beta-catenin was accompanied by a destabilization of complexes formed between these two proteins, leading to an enrichment of beta-catenin in the detergent-soluble fraction. Uninduced cells were able to form three-dimensional tubular structures in collagen I gels which were disrupted upon JunER activation, leading to irregular cell aggregates. The JunER-induced disruption of tubular structures was dependent on active signaling by growth factors. Moreover, the effects of JunER could be mimicked in normal cells by the addition of acidic fibroblast growth factor (aFGF). These data suggest that a possible function of c-Jun in epithelial cells is to modulate epithelial polarity and regulate tissue organization, processes which may be equally important for both normal breast development and as initiating steps in carcinogenesis.

The cadmium-induced disruption of tight junctions in LLC-PK 1 cells does not result from apoptosis

Exposure of LLC-PK 1 cells to low micromolar concentrations of Cd 2+ for 1–4 hours causes the disruption of the adhering and occluding junctions between the cells, whereas exposure to higher concentrations of Cd 2+ for longer periods of time causes more severe toxic effects and cell death. The objective of the present studies was to determine whether or not the junctional effects of Cd 2+ might be a consequence of apoptotic injury. LLC-PK 1 cells on cell culture inserts were exposed to either Cd 2+ or tumor necrosis factor (TNF-α) plus cycloheximide, a treatment that has recently been shown to cause apoptosis in LLC-PK 1 cells. The results showed that at the time the Cd 2+-induced junctional changes were occurring, there was no increase in the number of apoptotic cells or evidence of DNA fragmentation. By contrast, TNF-α plus cycloheximide induced changes that were characteristic of apoptosis. These results indicate that the disruption of intercellular junctions by Cd 2+ in the LLC-PK 1 cell line occurs independently of apoptosis.

Characterization of corneal endothelium cell cultured on microporous membrane filters.

In these experiments we characterize rabbit and bovine corneal endothelia cell cultured on microporous membrane filters (0.6cm2). Cell cultured bovine or rabbit corneal endothelial cells (subcultures 1-3) were seeded onto Millicell-HA filter inserts. Electrical resistance measured across the cultured monolayers increased steadily through 14 days of culture, reaching 34.2 +/- 0.8 ohm-cm2 (mean +/- SE) for rabbit cells and 33.1 +/- 1.1 ohm-cm2 for bovine cells. Alizarin red staining of the monolayers showed a polygonal morphology comparable to that observed in situ. Transmission electron microscopy showed well developed apical junctional complexes and flaps. Exposure of the monolayers to calcium-free medium resulted in the disruption of intercellular junctions, rounding-up of the cells and a decrease in electrical resistance (to near 0). Transmonolayer fluxes of inulin and dextran correlated well to the resistance measurements. Results of this study demonstrate that corneal endothelium, both bovine and rabbit, grown on filter inserts is comparable in morphology and ultrastructure to corneal endothelium in situ. The cells cultured in this system form functional apical junctional complexes that effect a barrier function comparable to that of the endothelium in situ.

Cytoskeletal control of centrioles movement during the establishment of polarity in Madin-Darby canine kidney cells.

The two centrioles that are localized close to each other and to the nucleus in single Madin-Darby Canine kidney cells (MDCK) move apart by distances as large as 13 microns after the establishment of extensive cellular junctions. Microfilaments, and possibly microtubules appear to be responsible for this separation. In fully polarized cells, the centrioles are localized just beneath the apical membrane. After disruption of intercellular junctions in low calcium medium, the centrioles move back towards the cell center. This process requires intact microtubules but happens even in the absence of microfilaments. These results indicate that the position of centrioles is determined by opposing forces produced by microtubules and microfilaments and suggest that the balance between these forces is modulated by the assembly of cellular junctions. Centriole separation appears to be an early event in the process that precedes their final positioning in the apical-most region of the polarized cell.

Effect of scatter factor on motility of epithelial cells and fibroblasts

The scatter factor is a protein released by fibroblasts that causes dispersal of epithelial cell colonies and disruption of intercellular junctions, as well as an alteration of morphology with ruffling and rapid extension and movement of pseudopodia. To find out if the scatter factor has a direct effect on cell migration, the Boyden chamber assay was used to determine the effect of partially purified factor on the migration of cells through 8 microns pore size filters. The results showed that the mobility of Madin-Darby canine kidney (MDCK) cells was stimulated, and usually maximal at 100 ng per ml, which is equivalent to 100 to 200 units of activity in the standard assay based on the morphology and arrangement of cells. The migration was due to chemotaxis and chemokinesis. A keratinocyte cell line was also sensitive as were, to a lesser extent, BSCl monkey kidney cells. The motility of freshly isolated fibroblasts and fibroblast cell lines, however, was not significantly affected. The results confirm the cell specificity and paracrine role of the scatter factor and show that this fibroblast-derived molecule can directly stimulate the movement of epithelial cells.

Comparative ultrastructural alterations in rabbit limb-buds after a teratogenic dose of either hydroxyurea or methotrexate.

Early changes in rabbit limb-bud fine structure subsequent to maternal administration of either hydroxyurea (750 mg HU/kg) or methotrexate (19.2 mg MTX/kg) on gestational day 12 have been analyzed in order to identify the morphologic sites of teratogenic action. Forelimb-buds were examined at 15 minutes to 32 hours posttreatment for ultrastructural alterations. HU caused early dispersion of ribosomes, condensation of nuclei, segregation of the granular portion of the nucleoli, and eventual fragmentation of mesenchymal cells. As a consequence of the extensive mesenchymal cellular death, the intercellular space among the remaining viable cells was increased. Cellular debris from fragmented cells was seen phagocytized within cells and in the intercellular space. In contrast, MTX caused an increase in cytoplasmic lipid, angular contours of nuclei, but relatively little cellular death. The most prominent change was a disruption of the normal mesenchymal architecture characterized by greatly increased intercellular space and a consequent reduction in the number of intercellular junctions. Nearly normal cytoarchitecture was restored by 32 hours. It was concluded that the primary morphologic sites of action are indeed different. Nevertheless, MTX and HU shared a common denominator since both caused disruption of intercellular contacts and possible breakdown of intercellular communication: HU through cytolethality, and MTX via disruption of intercellular junctions. This latter phenomenon may help to explain the similarities in limb defects caused by these two drugs.