Loss Of Transepithelial Resistance Research Articles

ER stress is associated with dedifferentiation and an epithelial-to-mesenchymal transition-like phenotype in PC Cl3 thyroid cells

Conditions perturbing the homeostasis of the endoplasmic reticulum (ER) cause accumulation of unfolded proteins and trigger ER stress. In PC Cl3 thyroid cells, thapsigargin and tunicamycin interfered with the folding of thyroglobulin, causing accumulation of this very large secretory glycoprotein in the ER. Consequently, mRNAs encoding BiP and XBP-1 were induced and spliced, respectively. In the absence of apoptosis, differentiation of PC Cl3 cells was inhibited. mRNA and protein levels of the thyroid-specific genes encoding thyroglobulin, thyroperoxidase and the sodium/iodide symporter and of the genes encoding the thyroid transcription factors TTF-1, TTF-2 and Pax-8 were dramatically downregulated. These effects were, at least in part, transcriptional. Moreover, they were selective and temporally distinct from the general and transient PERK-dependent translational inhibition. Thyroid dedifferentiation was accompanied by changes in the organization of the polarized epithelial monolayer. Downregulation of the mRNA encoding E-cadherin, and upregulation of the mRNAs encoding vimentin, alpha-smooth muscle actin, alpha(1)(I) collagen and SNAI1/SIP1, together with formation of actin stress fibers and loss of trans-epithelial resistance were found, confirming an epithelial-mesenchymal transition (EMT). The thyroid-specific and epithelial dedifferentiation by thapsigargin or tunicamycin were completely prevented by the PP2 inhibitor of Src-family kinases and by stable expression of a dominant-negative Src. Together, these data indicate that ER stress induces dedifferentiation and an EMT-like phenotype in thyroid cells through a Src-mediated signaling pathway.

Role of p38 MAP Kinase and Transforming Growth Factor-β Signaling in Transepithelial Migration of Invasive Bacterial Pathogens

Streptococcus pneumoniae and Haemophilus influenzae are human pathogens that often asymptomatically colonize the mucosal surface of the upper respiratory tract, but also occasionally cause invasive disease. The ability of these species to traverse the epithelium of the airway mucosa was modeled in vitro using polarized respiratory epithelial cells in culture. Migration across the epithelial barrier was preceded by loss of transepithelial resistance. Membrane products of S. pneumoniae that included lipoteichoic acid induced disruption of the epithelial barrier in a Toll-like receptor 2-dependent manner. This result correlates with a recent genetic study that associates increased TLR2 signaling with increased rates of invasive pneumococcal disease in humans. Loss of transepithelial resistance by the TLR2 ligand correlated with activation of p38 MAP kinase and transforming growth factor (TGF)-beta signaling. Activation of p38 MAPK and TGF-beta signaling in epithelial cells upon nasal infection with S. pneumoniae was also demonstrated in vivo. Inhibition of either p38 MAPK or TGF-beta signaling was sufficient to inhibit the migration of S. pneumoniae or H. influenzae. Our data shows that diverse bacteria utilize common mechanisms, including MAPK and TGF-beta signaling pathways to disrupt epithelial barriers and promote invasion.

RANTES mediates TNF-dependent lamina propria mast cell accumulation and barrier dysfunction in neurogenic cystitis

Barrier dysfunction of the urinary bladder is postulated to contribute to patient morbidity in the bladder inflammatory disease interstitial cystitis (IC). IC is often considered a neurogenic cystitis, but the mechanisms underlying barrier dysfunction are unclear. In murine neurogenic cystitis induced by pseudorabies virus (PRV), we previously observed formation of urothelial lesions characterized by urothelial apoptosis and urothelial discontinuities. Lesion formation was preceded by mast cell trafficking to the lamina propria, and trafficking was mediated by tumor necrosis factor-alpha (TNF). Here, we found that supernatants of TNF-treated urothelial cultures promoted chemotaxis of bone marrow-derived mast cells in vitro that was blocked by anti-RANTES antibodies but unaffected by anti-TNF antibodies. In vivo, PRV infection of wild-type mice induced RANTES expression in the urothelium that was temporally coincident with lamina propria mast cell accumulation (maximum at days 3-4 following infection) and was not induced in TNF(-/-) mice, TNFR1/2(-/-) mice, or mice treated with anti-TNF antibodies. Anti-RANTES antibodies blocked PRV-induced lamina propria mast cell accumulation 56% and reduced the prevalence of animals with detectable lesions 42%, relative to isotype control antibodies. Bladder barrier function was quantified by measuring transepithelial resistance (TER). PRV induced a 49% loss of TER in the presence of control antibodies, but mice treated with anti-RANTES antibodies exhibited reduced TER loss (16%, P < 0.01). These data demonstrate that RANTES plays a key role in the pathogenesis of neurogenic cystitis and suggest that chemokines may represent novel therapeutic targets for IC patients with mast cell-associated disease.

Bves modulates epithelial integrity through an interaction at the tight junction

We first identified Bves (blood vessel/epicardial substance) as a transmembrane protein that localized to the lateral compartment of the epithelial epicardium. Bves traffics to sites of cell-cell contact in cultured epicardial cells and promotes adhesion following transfection into non-adherent fibroblastic L-cells, reminiscent of a cell adhesion molecule. Currently, no function for Bves in relation to epithelial cell adhesion has been identified. We hypothesize that Bves plays a role at cell junctions to establish and/or modulate cell adhesion or cell-cell interactions in epithelial cell types. In this study, we demonstrate that Bves regulates epithelial integrity and that this function may be associated with a role at the tight junction (TJ). We report that Bves localizes with ZO-1 and occludin, markers of the TJ, in polarized epithelial cell lines and in vivo. We find that the behavior of Bves following low Ca2+ challenge or TPA treatment mimics that observed for ZO-1 and is distinct from adherens junction proteins such as E-cadherin. Furthermore, GST pull-down experiments show an interaction between ZO-1 and the intracellular C-terminal tail of Bves. Finally, we demonstrate that Bves modulates tight junction integrity, as indicated by the loss of transepithelial resistance and junction protein localization at the membrane following Bves knock-down in cultured cells. This study is the first to identify a function for Bves in epithelia and supports the hypothesis that Bves contributes to establishment and/or maintenance of epithelial cell integrity.

Enteropathogenic Escherichia coli outer membrane proteins induce changes in cadherin junctions of Caco-2 cells through activation of PKCα

Enteropathogenic Escherichia coli (EPEC) is a Gram-negative bacterial pathogen that adheres to human intestinal epithelial cells, resulting in watery, persistent diarrhoea. Despite the advances made in understanding EPEC–host cell interactions, the molecular mechanisms underlying watery diarrhoea have not been understood fully. Loss of transepithelial resistance and increased monolayer permeability by disruption of tight junctions has been implicated in this process. Apart from disruption of tight junctions, an important factor known to regulate monolayer permeability is E-cadherin and its interaction with β-catenin, both of which constitute the adherens junctions. Our previous studies using HEp-2 cells demonstrated the morphological and cytoskeletal changes caused by cell-free outer membrane preparations (OMPs) of EPEC. In this study, we have shown that EPEC and its OMP induce significant changes in the adherens junctions of Caco-2 monolayers. We also observed significant phosphorylation of protein kinase Cα (PKCα) in cells treated with either whole EPEC or its OMP. Immunoprecipitation of cell lysates with anti-E-cadherin and probing with phospho-PKCα monoclonal antibodies and anti-β-catenins revealed that in these cells, phosphorylated PKCα is associated with cadherins, leading to the dissociation of the cadherin/β-catenin complex. Immunofluorescence showed β-catenins dissociated from the membrane-bound cadherins and redistributed into the cytoplasm. Expression of dominant negative PKCα reversed these effects caused by either whole EPEC or its OMP and also reduced the associated increase in monolayer permeability. It is possible that this mechanism may complement the earlier known pathways for loss of barrier function involving myosin light chain kinase activation and also may play a role in causing host cell death by apoptosis.

Hepatocyte growth factor alters renal epithelial cell susceptibility to uropathogenic Escherichia coli.

The urinary tract is frequently the source of Escherichia coli bacteremia. Bacteria from the urinary tract must cross an epithelial layer to enter the bloodstream. Hepatocyte growth factor (HGF) alters the polarity of Madin-Darby canine kidney (MDCK) epithelial cells. The role of cell polarity in determining renal epithelial resistance to Escherichia coli invasion is not well known. A model of polarized and HGF-treated MDCK epithelial cells grown on filters was used to study the role of epithelial cell polarity during the interaction of nonvirulent (XL1-Blue) and uropathogenic (J96) strains of Escherichia coli with renal epithelium. Basolateral exposure of MDCK cells to J96, but not XL1-Blue, resulted in loss of transepithelial resistance (TER), which was due to epithelial cytotoxicity and not degradation of epithelial junctional proteins by bacterial proteases. Apical exposure to both J96 and XL1-Blue did not alter TER. Pretreatment of polarized MDCK cell monolayers with HGF renders the cells sensitive to loss of TER and cytotoxicity by apical exposure to J96. Analysis by confocal microscopy demonstrated that HGF treatment of MDCK cell monolayers also greatly enhances adherence of J96 to the apical surface of the cell monolayer. These data demonstrate that the basolateral surface of polarized epithelia is more susceptible to J96 cytotoxicity. The data also support the hypothesis that processes that alter epithelial cell polarity increase sensitivity of epithelia to bacterial injury and adherence from the apical compartment.

Reassembly of the Tight Junction after Oxidative Stress Depends on Tyrosine Kinase Activity

Oxidative stress compromises the tight junction, but the mechanisms underlying its recovery remain unclear. We developed a model in which oxidative stress reversibly disrupts the tight junction. Exposure of Madin-Darby canine kidney cells to hydrogen peroxide markedly reduced transepithelial resistance and disrupted the staining patterns of the tight junction proteins ZO-1 and occludin. These changes were reversed by catalase. The short-term reassembly of tight junctions was not dependent on new protein synthesis, suggesting that recovery occurs through re-utilization of existing proteins. Although ATP levels were reduced, the reduction was insufficient to explain the observed changes, since a comparable reduction of ATP levels (with 2-deoxy-D-glucose) did not induce these changes. The intracellular hydrogen peroxide scavenger pyruvate protected Madin-Darby canine kidney cells from loss of transepithelial resistance as did the heavy metal scavenger N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine. Of a wide variety of agents examined, only tyrosine kinase inhibitors and protein kinase C inhibitors markedly inhibited tight junction reassembly. During reassembly, tyrosine phosphorylation in or near the lateral membrane, was detected by immunofluorescence. The tyrosine kinase inhibitors genistein and PP-2 inhibited the recovery of transepithelial resistance and perturbed the relocalization of ZO-1 and occludin to the tight junction, indicating that tyrosine kinases, possibly members of the Src family, are critical for reassembly after oxidative stress.

Rotavirus alters paracellular permeability and energy metabolism in Caco-2 cells.

Rotaviruses infect epithelial cells of the small intestine, but the pathophysiology of the resulting severe diarrhea is incompletely understood. Histological damage to intestinal epithelium is not a consistent feature, and in vitro studies showed that intestinal cells did not undergo rapid death and lysis during viral replication. We show that rotavirus infection of Caco-2 cells caused disruption of tight junctions and loss of transepithelial resistance (TER) in the absence of cell death. TER declined from 300 to 22 Omega. cm(2) between 8 and 24 h after infection and was accompanied by increased transepithelial permeability to macromolecules of 478 and 4,000 Da. Distribution of tight junction proteins claudin-1, occludin, and ZO-1 was significantly altered during infection. Claudin-1 redistribution was notably apparent at the onset of the decline in TER. Infection was associated with increased production of lactate, decreased mitochondrial oxygen consumption, and reduced cellular ATP (60% of control at 24 h after infection), conditions known to reduce the integrity of epithelial tight junctions. In conclusion, these data show that rotavirus infection of Caco-2 intestinal cells altered tight junction structure and function, which may be a response to metabolic dysfunction.

Gastrointestinal glutathione peroxidase prevents transport of lipid hydroperoxides in CaCo-2 cells

Background & Aims: Gastrointestinal glutathione peroxidase (GI-GPx), 1 of the 4 types of selenium-dependent glutathione peroxidases, is expressed exclusively in the gastrointestinal system and has therefore been suggested to function as a barrier against the absorption of dietary hydroperoxides. Methods: The selenium-dependent expression of GI-GPx and cytosolic GPx (cGPx) was analyzed by Western blotting. Transport of 13-hydroperoxy octadecadienoic acid (13-HPODE) was investigated in a CaCo-2 cell monolayer modulated in GI-GPx and cGPx by selenium restriction or repletion. Localization of GI-GPx in rat intestine was visualized by immunohistochemistry. Results: Low but significant GI-GPx levels were detected in selenium-deficient CaCo-2 cells and in the gastrointestinal tract of selenium-deficient rats, whereas cGPx was completely absent. Selenium supplementation of CaCo-2 cells resulted in a 5-fold increase of GI-GPx protein, whereas total GPx activity increased by a factor of 13, with most of the GPx activity under selenium-adequate conditions being cGPx. Irrespective of the selenium status, 13-HPODE did not reach the basolateral side of an intact CaCo-2 cell monolayer. Depending on the selenium status, hydroperoxides damaged the monolayer as evidenced by loss of transepithelial resistance and paracellular diffusion of lucifer yellow. Only under these conditions was unmetabolized 13-HPODE detectable at the basolateral side. Conclusions: Low GI-GPx levels, as present in selenium deficiency, suffice to prevent transport of 13-HPODE. GI-GPx may thus function as a barrier against hydroperoxide absorption. cGPx contributes to balance major oxidative challenge.GASTROENTEROLOGY 2000;119:420-430

CO-CULTURE OF HUMAN MONOCYTES AND THYROCYTES IN BICAMERAL CHAMBER: MONOCYTE-DERIVED IL-1α IMPAIRS THE THYROID EPITHELIAL BARRIER

Pro-inflammatory cytokines are important mediators in tissue responses to a wide range of endogenous (e.g. autoantigens) and exogenous (e.g. infections, wounds, biomaterials) stimuli. The complex interactions taking place between different cell types in such processes are difficult to examine in vivo. Here we studied the effect of human monocytes on thyroid epithelial cells co-cultured in bicameral chambers. Freshly isolated monocytes (1×106/ml) added to the basal compartment reduced the transepithelial resistance (from 300–600 to <100Ω·cm2) and caused a disruption of the tight junctions in apically grown thyrocyte monolayers after co-culture for 24h. The barrier function was further attenuated by monocytes exposed to lipopolysaccharide (10μg/ml) or polystyrene microspheres (size: 3μm; 1×107/ml). Loss of transepithelial resistance was accompanied by release of interleukin 1α (maximally 550pg/ml) from the monocytes. Conversely, the resistance remained high when co-cultures were simultaneously incubated with neutralizing anti-human interleukin 1α antibodies. The results show that the integrity of cultured thyroid epithelium is impaired by monocytes without requirement of direct cell-to-cell contact. This action, mediated by interleukin-1α, suggests a mechanism by which hidden (lumenal) autoantigens might be exposed to interstitial antigen-presenting cells in autoimmune thyroid disease. In perspective, the model provides a tool in which humoral and cell-cell dependent processes generated by bioactive agents and particulate materials, for instance, during the healing and repair of tissue around biomaterials and hybrid implants, can be selectively examined.

Protein Kinase C-α Activity Modulates Transepithelial Permeability and Cell Junctions in the LLC-PK1 Epithelial Cell Line

Modulation of protein kinase C (PKC) by 12-O-tetradecanoylphorbol-13-acetate (TPA) disrupts the cell-cell junctions of the epithelial cell line LLC-PK1. To examine the role of specific PKC isoforms in this process we have created modified LLC-PK1 subclones that express wild-type and dominant negative versions of PKC-alpha under control of the tetracycline-responsive expression system. Overexpression of wild-type PKC-alpha rendered the cells more sensitive to the effects of TPA on transepithelial permeability as measured by loss of transepithelial resistance across the cell sheet. Conversely, expression of a dominant negative PKC-alpha rendered the cells more resistant to the effects of TPA as measured both by loss of transepithelial resistance as well as cell scattering. The properties of both subclones could be modulated by the addition of tetracycline, which suppressed the effect of the exogenous genes. These results indicate that the alpha isoform of PKC is at least one of the isoforms that regulate tight junctions and other cell-cell junctions of LLC-PK1 epithelia.

Activation of a transfected FGFR-1 receptor in Madin-Darby epithelial cells results in a reversible loss of epithelial properties.

Basic fibroblast growth factor (bFGF) is a potent mitogen for a wide variety of cell types derived from mesoderm and neuroectoderm. The activity of bFGF is mediated by several types of closely related receptors belonging to the tyrosine-kinase family of receptors. We have found that Madin-Darby epithelial cells (MDCK) do not seem to produce bFGF or bFGF receptors. High level expression of human bFGF cDNA in these cells did not produce any mitogenic or morphological effects. Expression of the mouse-derived cDNA encoding FGF receptor-1 (FGFR-1) in MDCK cells resulted in the acquisition of a fibroblast-like morphology when the transfected cells were cultured at low density in the presence of 0.6% fetal calf serum and 20 ng/ml bFGF. Acidic fibroblast growth factor (aFGF) also induced these morphological changes but not keratinocyte growth factor. The morphological effect was not accompanied by increased bFGF-induced cell proliferation and did not result in the loss of epithelial cell markers such as cytokeratins. However, the morphological transition was accompanied by changes in the intracellular distribution of actin. In spite of these changes the transfected cells formed monolayers even in the presence of bFGF. Coexpression of bFGF and FGFR-1 in the MDCK cells resulted in similar morphological effects that were not dependent upon exogenous bFGF. These morphological effects were mimicked by exposure of MDCK cells to either orthovanadate or phorbol ester. Parental and FGFR-1-expressing MDCK cells formed monolayers that displayed high electrical resistance. Incubation of monolayers of FGFR-1-transfected cells with bFGF resulted in the loss of trans-epithelial resistance. Monolayers of parental MDCK cells did not lose their trans-epithelial resistance in response to bFGF, although exposure to phorbol ester did result in the loss of their trans-epithelial resistance, indicating that the effects on the trans-epithelial resistance are mediated by protein kinase C activation. Interestingly, orthovanadate did not cause a loss of transepithelial resistance, suggesting that the loss of trans-epithelial resistance is separable from the morphological transition.

Salmonella Interactions with Polarized Human Intestinal Caco-2 Epithelial Cells

Polarized monolayers of the human intestinal epithelial Caco-2 cell line were grown on permeable filters and infected apically with either Salmonella choleraesuis or Salmonella typhimurium. Both Salmonella species penetrated through the monolayer, requiring 2 h before appearing in the basolateral medium. Both species caused a loss in transepithelial resistance by 3-4 h, and the monolayer's integrity was completely disrupted by 6 h. Scanning and transmission electron microscopy revealed that the bacteria interacted with well-defined apical microvilli and caused disruptions in the brush border, including elongation and denuding of the microvilli. The cytoplasm was also disrupted locally, with blebs protruding from the apical surface. The bacteria entered (invaded) these cells and were enclosed in membrane-bound vacuoles within the cytoplasm. By 6 h there were many bacteria within most Caco-2 cells, and these organisms caused serious cytopathic consequences. These morphologic observations correlated well with animal infection models, indicating that this in vitro system will be useful to study pathogens that interact with human intestinal epithelia.