Formation of tight junctions in epithelial cells. I. Induction by proteases in a human colon carcinoma cell line

The experimental modulation of tight junctions (TJ) was studied in the human adenocarcinoma cell line HT 29 by freeze-fracture electron microscopy. The cell line has virtually no TJ when grown in culture. TJ could be induced by mild treatment with a variety of endopeptidases (trypsin, chymotrypsin, collagenase, elastase, plasmin, thrombin, papain, and pronase). Pronase induced the formation of TJ at low (but not at high) concentrations. All exopeptidases studied were unable to induce the formation of TJ. At 0 degree C the trypsin-induced formation of TJ was greatly slowed down although not entirely inhibited. However, when cells were briefly treated with trypsin at 0 degree C and subsequently transferred to 37 degrees C in the presence of protease inhibitors, TJ were rapidly assembled. Thus an induction phase at low temperature and an assembly phase at high temperature could be experimentally separated. When cells were briefly trypsinized at 0 degrees and subsequently kept at 0 degree C without trypsin for several hours, TJ still formed abundantly upon incubation at 37 degrees C. It appears therefore that the effect produced by the protease is retained for long periods in the cold.     

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.     

Assessment of tight junctions between pulmonary epithelial and endothelial cells

This study is intended to determine whether qualitative assessment of tight junction integrity from freeze-fracture data is reliable. We used lung parenchyma from a control mongrel dog's cardiac lung lobe, from a mongrel dog subjected to vascular high-pressure pulmonary edema (HPPE), and from a dog subjected to oleic acid-induced low-pressure pulmonary edema (LPPE) (6). Quantitative assessment was done on 115 freeze-fracture micrographs of epithelial tight junctions and on another 158 freeze-fracture micrographs of endothelial junctions from the 3 dogs. Quantitative assessment showed differences between the dogs in junction depth, fibril numbers, density, and complexity. for qualitative assessment, these same 273 micrographs were assessed in a single-blind fashion by having six investigators sort first the epithelial and then the endothelial junctions into normal or damaged categories. Qualitative assessment did not agree with quantitative data, suggesting that it is unreliable.     

Role of tight junctions in cell proliferation and cancer

The acquisition of a cancerous phenotype by epithelial cells involves the disruption of intercellular adhesions. The reorganization of the E-cadherin/beta-catenin complex in adherens junctions during cell transformation is widely recognized. Instead the implication of tight junctions (TJs) in this process is starting to be unraveled. The aim of this article is to review the role of TJ proteins in cell proliferation and cancer.     

Ultrastructural characterization of two new human endometrial carcinoma cell lines and normal human endometrial epithelial cells cultured on extracellular matrix

Summary Two new lines of human endometrial carcinoma (HEC) cells, one from an adenocarcinoma and one from a highly metastatic serous papillary carcinoma, were established in culture. Structural and morphologic properties of these cells at early passage were compared with those of cultured normal human endometrial epithelial (NHEE) cells. For these studies, cells were grown on a conventional plastic surface or on an extracellular matrix substrate (Matrigel), and examined by transmission electron microscopy and immunofluorescent light microscopy. The HEC cells appeared morphologically similar on plastic and Matrigel, whereas the NHEE cells showed significantly greater epithelial morphologic differentiation on Matrigel than on plastic. On extracellular matrix, the morphologic differences observed between HEC cells and NHEE cells were primarily of an architectural nature, which may be in part explained by differences between NHEE and HEC cells in the arrangement of actin microfilaments and cytokeratin intermediate filaments. Furthermore, HEC cells displayed extensive networks of vimentin intermediate filaments, which were absent from the NHEE cells. These observations support the hypothesis that architectural deregulation is a prominent feature of endometrial carcinoma, and that cytoskeletal alterations may uncouple HEC cell ultrastructural morphology from the influence of extracellular matrix. This research was supported by research grants CA31733, CA45727, and ES07017, from the National Institutes of Health, Bethesda, MD. G. P. S. is a Jefferson Pilot Fellow in Academic Medicine. A preliminary account of this work was presented at the 1988 U.S.-Canadian Academy of Pathology Annual Meeting (Lab. Inves. 58:12a, 1988).     

Regulation of airway tight junctions by proinflammatory cytokines

Epithelial tight junctions (TJs) provide an important route for passive electrolyte transport across airway epithelium and provide a barrier to the migration of toxic materials from the lumen to the interstitium. The possibility that TJ function may be perturbed by airway inflammation originated from studies reporting (1) increased levels of the proinflammatory cytokines interleukin-8 (IL-8), tumor necrosis factor alpha (TNF-alpha), interferon gamma (IFN-gamma), and IL-1beta in airway epithelia and secretions from cystic fibrosis (CF) patients and (2) abnormal TJ strands of CF airways as revealed by freeze-fracture electron microscopy. We measured the effects of cytokine exposure of CF and non-CF well-differentiated primary human airway epithelial cells on TJ properties, including transepithelial resistance, paracellular permeability to hydrophilic solutes, and the TJ proteins occludin, claudin-1, claudin-4, junctional adhesion molecule, and ZO-1. We found that whereas IL-1beta treatment led to alterations in TJ ion selectivity, combined treatment of TNF-alpha and IFN-gamma induced profound effects on TJ barrier function, which could be blocked by inhibitors of protein kinase C. CF bronchi in vivo exhibited the same pattern of expression of TJ-associated proteins as cultures exposed in vitro to prolonged exposure to TNF-alpha and IFN-gamma. These data indicate that the TJ of airway epithelia exposed to chronic inflammation may exhibit parallel changes in the barrier function to both solutes and ions.     

Maintenance of the epithelial barrier in a bronchial epithelial cell line is dependent on functional E-cadherin local to the tight junctions

Tight junctions (TJ) are essential components of polarized epithelia, and E-cadherin is important for their formation and maintenance. The bronchial epithelial cell line, 16HBE14o- expresses E- and P-cadherin, but not N-cadherin. E- and P-cadherin levels changed during culture, the former increasing after confluence, and the latter were markedly reduced. All detectable E-cadherin was bound to β- and γ-catenins. We investigated involvement of E-cadherin with epithelial integrity using an E-cadherin specific, function-blocking antibody, SHE78-7. Surprisingly, apical SHE78-7 exposure caused a prompt fall in transepithelial resistance (TER), while TER remained unchanged for 8 hrs after basal exposure then dropped. SHE78-7 exposure increased epithelial permeability to mannitol, inulin, and 9.5 kDa and 77 kDa dextrans and caused fragmentation and loss of the tight junction protein, ZO-1, from the cell borders in some areas. Ultrastructural studies showed that all junctional intercellular contact was lost in the center of SHE78-7 induced lesions. Near the lesion periphery, epithelial structure was maintained, but TJs were dysfunctional as shown by ruthenium red penetration. Analysis of epithelial penetration by SHE78-7 revealed discrete, local defects in the apical barrier at the top of some cell hills that permitted rapid access of the antibody to E-cadherin near the apical surface. In contrast, after basal exposure, antibody initially engaged with E-cadherin nearer the basal surface and only accessed apical E-cadherin later. Taken together with the TER measurements, these data suggest compartmentalization of E-cadherin function within 16HBE14o- cells, with only the apical E-cadherin adjacent to the tight junctions contributing to the function of the latter.     

Freeze-fracture replica studies of tight junctions in normal human bronchial epithelium

Using freeze-fracture techniques, tight junctional networks were observed in the human normal bronchial epithelium. They were morphologically classified into three types: type I was a loosely interconnected, most complicated network consisting of 7-11 roughly parallel wavy strands and situated between ciliated cells; type II was a randomly anastomosing, simple network made up of 2-4 strands and present between goblet cells; type III was an irregularly anastomosing network composed of 4-7 strands and located between a ciliated cell and a goblet cell. Type III junctions, when a goblet cell was strongly bulged, were located on the swollen ridge, the upper surface of which was separated by a deep groove from the bulged apical surface, around the lateral surface of the cell at the level of the luminal surface. The possible relation between the orientation of strands of these networks and extra- or intracellular stress was discussed.     

Regulation of SBP synthesis in human cancer cell lines by steroid and thyroid hormones

The presence of human sex steroid binding protein (SBP) in liver cells, the supposed site of SBP synthesis, and in other target cells for sex steroid hormones such as breast, endometrium and prostate epithelium, have been demonstrated by indirect immunofluorescence. It is not known whether SBP enters endometrial and prostate cells by endocytosis, possibly mediated by a cell membrane receptor process, or if SBP is synthesized in these cells. SBP mRNA has been searched in human cancer cell lines originated from liver (Hep G2/H5A), breast (MCF-7), endometrium (RL95-2) and prostate (LNCaP). It was only found in hepatoma cells where it is regulated by estradiol, antiestrogen tamoxifen and triiodothyronine, in a similar way as secreted SBP. This work provides evidence that human SBP is synthesized in the liver, and it also suggests that its regulation may involve non-steroidal factors.     

Complex tight junctions of epithelial and of endothelial cells in early foetal brain

Brain Cell Biology - The morphology of epithelial and of endothelial intercellular junctions in human foetal (9–15 weeks gestation) and sheep foetal (50, 60 and 125 days gestation, term 147...     

Salmonella type III effector AvrA stabilizes cell tight junctions to inhibit inflammation in intestinal epithelial cells

Salmonella Typhimurium is a major cause of human gastroenteritis. The Salmonella type III secretory system secretes virulence proteins, called effectors. Effectors are responsible for the alteration of tight junction (TJ) structure and function in intestinal epithelial cells. AvrA is a newly described bacterial effector found in Salmonella. We report here that AvrA expression stabilizes cell permeability and tight junctions in intestinal epithelial cells. Cells colonized with an AvrA-deficient bacterial strain (AvrA-) displayed decreased cell permeability, disruption of TJs, and an increased inflammatory response. Western blot data showed that TJ proteins, such as ZO-1, claudin-1, decreased after AvrA- colonization for only 1 hour. In contrast, cells colonized with AvrA-sufficient bacteria maintained cell permeability with stabilized TJ structure. This difference was confirmed in vivo. Fluorescent tracer studies showed increased fluorescence in the blood of mice infected with AvrA- compared to those infected with the AvrA-sufficient strains. AvrA- disrupted TJ structure and function and increased inflammation in vivo, compared to the AvrA- sufficient strain. Additionally, AvrA overexpression increased TJ protein expression when transfected into colonic epithelial cells. An intriguing aspect of this study is that AvrA stabilized TJs, even though the other TTSS proteins, SopB, SopE, and SopE2, are known to disrupt TJs. AvrA may play a role in stabilizing TJs and balancing the opposing action of other bacterial effectors. Our findings indicate an important role for the bacterial effector AvrA in regulation of intestinal epithelial cell TJs during inflammation. The role of AvrA represents a highly refined bacterial strategy that helps the bacteria survive in the host and dampen the inflammatory response.     

TNFAIP3 maintains intestinal barrier function and supports epithelial cell tight junctions

Tight junctions between intestinal epithelial cells mediate the permeability of the intestinal barrier, and loss of intestinal barrier function mediated by TNF signaling is associated with the inflammatory pathophysiology observed in Crohn's disease and celiac disease. Thus, factors that modulate intestinal epithelial cell response to TNF may be critical for the maintenance of barrier function. TNF alpha-induced protein 3 (TNFAIP3) is a cytosolic protein that acts in a negative feedback loop to regulate cell signaling induced by Toll-like receptor ligands and TNF, suggesting that TNFAIP3 may play a role in regulating the intestinal barrier. To investigate the specific role of TNFAIP3 in intestinal barrier function we assessed barrier permeability in TNFAIP3(-/-) mice and LPS-treated villin-TNFAIP3 transgenic mice. TNFAIP3(-/-) mice had greater intestinal permeability compared to wild-type littermates, while villin-TNFAIP3 transgenic mice were protected from increases in permeability seen within LPS-treated wild-type littermates, indicating that barrier permeability is controlled by TNFAIP3. In cultured human intestinal epithelial cell lines, TNFAIP3 expression regulated both TNF-induced and myosin light chain kinase-regulated tight junction dynamics but did not affect myosin light chain kinase activity. Immunohistochemistry of mouse intestine revealed that TNFAIP3 expression inhibits LPS-induced loss of the tight junction protein occludin from the apical border of the intestinal epithelium. We also found that TNFAIP3 deubiquitinates polyubiquitinated occludin. These in vivo and in vitro studies support the role of TNFAIP3 in promoting intestinal epithelial barrier integrity and demonstrate its novel ability to maintain intestinal homeostasis through tight junction protein regulation.     

Selected polymorphisms in sex hormone-related genes, circulating sex hormones and risk of endometrial cancer

Background: The role of estrogen and progesterone in the development of endometrial cancer is well documented. Few studies have examined the association of genetic variants in sex hormone-related genes with endometrial cancer risk. Methods: We conducted a case-control study nested within three cohorts to examine the association of endometrial cancer risk with polymorphisms in hormone-related genes among 391 cases (92% postmenopausal at diagnosis) and 712 individually-matched controls. We also examined the association of these polymorphisms with circulating levels of sex hormones and SHBG in a cross-sectional analysis including 596 healthy postmenopausal women at blood donation (controls from this nested case-control study and from a nested case-control study of breast cancer in one of the three cohorts). Results: Adjusting for endometrial cancer risk factors, the A allele of rs4775936 in CYP19 was significantly associated (OR(per allele)=1.22, 95% CI=1.01-1.47, p(trend)=0.04), while the T allele of rs10046 was marginally associated with increased risk of endometrial cancer (OR(per allele)=1.20, 95% CI=0.99-1.45, p(trend)=0.06). PGR rs1042838 was also marginally associated with risk (OR(per allele)=1.25, 95% CI=0.96-1.61, p(trend)=0.09). No significant association was found for the other polymorphisms, i.e. CYP1B1 rs1800440 and rs1056836, UGT1A1 rs8175347, SHBG rs6259 and ESR1 rs2234693. Rs8175347 was significantly associated with postmenopausal levels of estradiol, free estradiol and estrone and rs6259 with SHBG and estradiol. Conclusion: Our findings support an association between genetic variants in CYP19, and possibly PGR, and risk of endometrial cancer.     

Molecular physiology and pathophysiology of tight junctions. IV. Regulation of tight junctions by extracellular stimuli: nutrients, cytokines, and immune cells

The epithelial lining of the gastrointestinal tract forms a regulated, selectively permeable barrier between luminal contents and the underlying tissue compartments. Permeability across the epithelium is, in part, determined by the rate-limiting barrier of the paracellular pathway-the most apical intercellular junction referred to as the tight junction (TJ). The TJ is composed of a multiprotein complex that affiliates with the underlying apical actomyosin ring. TJ structure and function, and therefore epithelial permeability, are influenced by diverse physiological and pathological stimuli; here we review examples of such stimuli that are detected at the cell surface. For example, luminal glucose induces an increase in paracellular permeability to small molecules. Similarly, but by other means, cytokines and leukocytes in the vicinity of the epithelium also regulate TJ structure and paracellular permeability by influencing the TJ protein complex and/or its association with the underlying actin cytoskeleton.     

Caprine endometrial stromal cells modulate the effects of steroid hormones on cytokine secretion by endometrial epithelial cells in vitro

The main purpose of this study was to examine the effects of 17β-estradiol (E₂) and progesterone (P₄) on cytokine secretion by caprine endometrial epithelial cells (EEC) in vitro. Epithelial cells grown alone or in co-culture with stromal cells (ESC) were treated with E₂ or P₄, or both. Homogeneity of the endometrial cell populations was ascertained immunocytochemically. The quantities of cytokines secreted in this system were assessed by ELISA and their protein expression by Western blot. The exposure of EEC to P₄ alone or in combination with E₂ significantly increased the amount of TGF-β1, TNF-α and IL-18 secretion, whereas E₂ had no effect on the synthesis of these cytokines. When epithelial cells were co-cultured with ESC, the secretion of TGF-β1, TNF-α and IL-18 by EEC significantly increased compared to that by EEC alone. However, the treatment with both steroids decreased the secretion of TNF-α, IL-18 and TGF-β1 by EEC in the presence of ESC. In contrast to TGF-β1, TNF-α and IL-18, the secretion of leukemia inhibitory factor (LIF) by EEC was not affected by E₂ and/or P₄ either directly or indirectly. The present results indicate that the interactions between caprine endometrial stromal and epithelial cells can modulate the secretion of TGF-β1, TNF-α and IL-18 by EEC exposed to E₂ and/or P₄ in vitro.     

Contribution of claudin-5 to barrier properties in tight junctions of epithelial cells

Claudin-5 is a transmembrane protein reported to be primarily present in tight junctions of endothelia. Unexpectedly, we found expression of claudin-5 in HT-29/B6 cells, an epithelial cell line derived from human colon. Confocal microscopy showed colocalization of claudin-5 with occludin, indicating its presence in the tight junctions. By contrast, claudin-5 was absent in the human colonic cell line Caco-2 and in Madin-Darby canine kidney cells (MDCK sub-clones C7 and C11), an epithelial cell line derived from the collecting duct. To determine the contribution of claudin-5 to tight junctional permeability in cells of human origin, stable transfection of Caco-2 with FLAG-claudin-5 cDNA was performed. In addition, clone MDCK-C7 was transfected. Synthesis of the exogenous FLAG-claudin-5 was verified by Western blot analysis and confocal fluorescent imaging by employing FLAG-specific antibody. FLAG-claudin-5 was detected in transfected cells in colocalization with occludin, whereas cells transfected with the vector alone did not exhibit specific signals. Resistance measurements and mannitol fluxes after stable transfection with claudin-5 cDNA revealed a marked increase of barrier function in cells of low genuine transepithelial resistance (Caco-2). By contrast, no changes of barrier properties were detected in cells with a high transepithelial resistance (MDCK-C7) after stable transfection with claudin-5 cDNA. We conclude that claudin-5 is present in epithelial cells of colonic origin and that it contributes to some extent to the paracellular seal. Claudin-5 may thus be classified as a tight-junctional protein capable of contributing to the "sealing" of the tight junction.     

Molecular physiology and pathophysiology of tight junctions. I. Biogenesis of tight junctions and epithelial polarity

The tight junction (TJ) was first noticed through its ability to control permeation across the paracellular route, but the homologies of its molecular components with peptides that participate in tumor suppression, nuclear addressing, and cell proliferation indicate that it may be involved in many other fundamental functions. TJs are formed by a dozen molecular species that assemble through PDZ and other protein-protein clustering promoting sequences, in response to the activation of E-cadherin. The TJ occupies a highly specific position between the apical and the basolateral domains. Its first molecular components seem to be delivered to such a position by addressing signals in their molecule and, once anchored, serve as a clustering nucleus for further TJ-associated molecules. Although in mature epithelial cells TJs and E-cadherin do not colocalize, a complex chain of reactions goes from one to the other that involves alpha-, beta-, and gamma-catenins, two different G proteins, phospholipase C, protein kinase C, calmodulin, mitogen-activated protein kinase, and molecules pertaining to the cytoskeleton, which keep the TJ sensitive to physiological requirements and local conditions (notably to Ca(2+)-dependent cell-cell contacts) throughout the life of the epithelium.