Zonula occludens-1 and -2 regulate apical cell structure and the zonula adherens cytoskeleton in polarized epithelia

The structure and function of both adherens (AJ) and tight (TJ) junctions are dependent on the cortical actin cytoskeleton. The zonula occludens (ZO)-1 and -2 proteins have context-dependent interactions with both junction types and bind directly to F-actin and other cytoskeletal proteins, suggesting ZO-1 and -2 might regulate cytoskeletal activity at cell junctions. To address this hypothesis, we generated stable Madin-Darby canine kidney cell lines depleted of both ZO-1 and -2. Both paracellular permeability and the localization of TJ proteins are disrupted in ZO-1/-2-depleted cells. In addition, immunocytochemistry and electron microscopy revealed a significant expansion of the perijunctional actomyosin ring associated with the AJ. These structural changes are accompanied by a recruitment of 1-phosphomyosin light chain and Rho kinase 1, contraction of the actomyosin ring, and expansion of the apical domain. Despite these changes in the apical cytoskeleton, there are no detectable changes in cell polarity, localization of AJ proteins, or the organization of the basal and lateral actin cytoskeleton. We conclude that ZO proteins are required not only for TJ assembly but also for regulating the organization and functional activity of the apical cytoskeleton, particularly the perijunctional actomyosin ring, and we speculate that these activities are relevant both to cellular organization and epithelial morphogenesis.     

Protease inhibitors suppress the formation of tight junctions in gastrointestinal cell lines.

Tight junctions (TJ) of the fascia occludens type can be induced in the human colon adenocarcinoma cell lines HT29 and Caco-2 by treatment with 320 mM cesium sulfate. This process can be completely inhibited by the protease inhibitors leupeptin and antipain. The concentration for 50% inhibition was 32 microM leupeptin and 270 microM antipain, respectively. In the polarized colon carcinoma cell line Caco-2, the spontaneous formation of histotypical TJ and the development of transepithelial electrical resistance do not occur when the cells are cultured in medium containing 400 microM leupeptin. Following the removal of leupeptin, zonula occludens type TJ and electrical resistance develop synchronously during a period of 4 h. Dihydroleupeptin, the alcohol analog of leupeptin, inhibits neither the spontaneous nor the induced assembly of TJ fibrils. Thus, the aldehyde group of leupeptin is essential for activity. These data suggest that the salt-induced as well as the spontaneous formation of TJ involve cellular proteases which are susceptible to protease inhibitors.     

Occludin-deficient Embryonic Stem Cells Can Differentiate into Polarized Epithelial Cells Bearing Tight Junctions

Occludin is the only known integral membrane protein of tight junctions (TJs), and is now believed to be directly involved in the barrier and fence functions of TJs. Occludin-deficient embryonic stem (ES) cells were generated by targeted disruption of both alleles of the occludin gene. When these cells were subjected to suspension culture, they aggregated to form simple, and then cystic embryoid bodies (EBs) with the same time course as EB formation from wild-type ES cells. Immunofluorescence microscopy and ultrathin section electron microscopy revealed that polarized epithelial (visceral endoderm-like) cells were differentiated to delineate EBs not only from wild-type but also from occludin-deficient ES cells. Freeze fracture analyses indicated no significant differences in number or morphology of TJ strands between wild-type and occludin-deficient epithelial cells. Furthermore, zonula occludens (ZO)-1, a TJ-associated peripheral membrane protein, was still exclusively concentrated at TJ in occludin-deficient epithelial cells. In good agreement with these morphological observations, TJ in occludin-deficient epithelial cells functioned as a primary barrier to the diffusion of a low molecular mass tracer through the paracellular pathway. These findings indicate that there are as yet unidentified TJ integral membrane protein(s) which can form strand structures, recruit ZO-1, and function as a barrier without occludin.     

Tight function zonula occludens-3 regulates cyclin D1-dependent cell proliferation

Coordinated regulation of cell proliferation is vital for epithelial tissue homeostasis, and uncontrolled proliferation is a hallmark of carcinogenesis. A growing body of evidence indicates that epithelial tight junctions (TJs) play a role in these processes, although the mechanisms involved are poorly understood. In this study, we identify and characterize a novel plasma membrane pool of cyclin D1 with cell-cycle regulatory functions. We have determined that the zonula occludens (ZO) family of TJ plaque proteins sequesters cyclin D1 at TJs during mitosis, through an evolutionarily conserved class II PSD-95, Dlg, and ZO-1 (PDZ)-binding motif within cyclin D1. Disruption of the cyclin D1/ZO complex through mutagenesis or siRNA-mediated suppression of ZO-3 resulted in increased cyclin D1 proteolysis and G(0)/G(1) cell-cycle retention. This study highlights an important new role for ZO family TJ proteins in regulating epithelial cell proliferation through stabilization of cyclin D1 during mitosis.     

A study of metabolic cooperation with established myoblast cell lines.

The present study was undertaken to test the action of ConA on the distribution of intramembranous particles (IMPs) and on the reassembly of junctional contacts in isolated and reaggregated embryonic neuronal and glial cells. The lectin ConA causes all embryonic cells to aggregate in unorganized cell patterns. ConA does not alter the distribution of IMPs but it inhibits the formation of the zonula occludens (ZO) by preventing the alignment and fusion of IMPs or by inducing them to become arranged in bizarre arrays. The possible relationship between ConA receptor sites and the IMPs is discussed. From a morphological viewpoint the aggregation of embryonic cells influenced by lectin is distinctly different from the normal processes of cell adhesion, cell sorting and establishment of intercellular contacts.     

Electron microscopic study of the intercellular junctions during the ciliary epithelial differentiation of the eye of the common frog]

Intercellular junctions in the eye ciliary epithelium were studied in Rana temporaria by means of transmission electron microscopy beginning from the stage of appearance of the light sensitivity in the larval eye and until the completion of metamorphosis. In the ciliary epithelium inner layer the apical parts of adjacent cells are tied by contacts forming the junction complex already at a very early developmental stage. The most apical position in this complex is occupied by the focal junction which appears to be an early stage of zonula occludens; this complex includes also zonula adherens and macula adherens which may follow in any succession focal junction or zonula occludens. No definite order was found in the localization of cell contacts between the outer and inner layers of ciliary epithelium, as well as between the side surfaces of the cells within each layer. Contacts were found everywhere termed as "lengthy" which may be considered as gap junctions. Regional differences were found in the ultrastructure of the ciliary epithelium cells with respect to unequal distribution of functional loads.     

Molecular composition of tight and adherens junctions in the rat olfactory epithelium and fila

Tight and adherens junctions (TJs, AJs) between neurons, epithelial and glial cells provide barrier and adhesion properties in the olfactory epithelium (OE), and subserve functions such as compartmentalization and axon growth in the fila olfactoria (FO). Immunofluorescence and immunoelectronmicroscopy were combined in sections of rat OE and FO to document the cellular and subcellular localization of TJ proteins occludin(Occl), claudins(Cl) 1-5 and zonula occludens(ZO) proteins 1-3, and of AJ proteins N-cadherin(cad), E-cad, and alpha-, beta- and p120-catenin(cat). With the exception of Cl2, all TJ proteins were colocalized in OE junctions. Differences in relative immunolabeling intensities were noted between neuronal and epithelial TJs. In the FO, Cl5-reactivity was localized in olfactory ensheathing cell (OEC) junctions, Cl1-reactivity in the FO periphery, with differential colocalization with ZOs. Supporting cells formed N-cad-immunoreactive (ir) AJs with olfactory sensory neurons, E-cad-ir junctions with microvillar and gland duct cells, and both N-cad and E-cad-ir junctions in homotypic contacts. Alpha, beta- and p120-cat were localized in all AJs of the OE. AJs were scarce in the globose basal cell layer. Immature and mature neurons formed numerous contacts. In the FO, AJs were documented between OECs, between OECs and axons, and between axons. Most AJs colocalized N-cad with catenins, occasionally E-cad-ir AJs were found in the FO periphery. Characteristics of molecular composition suggest differential properties of TJs formed by neuronal, epithelial and glial cells in the OE and FO. The presence and molecular composition of AJs are consistent with a role of AJ proteins in neuroplastic processes in the peripheral olfactory pathway.     

Cell junctions in the renal tubule of a fresh-water teleost,Salmo gairdneri Rich.

Summary Cell junctions in the renal tubule of the fresh-water rainbow trout were studied with thin-section and freeze-fracture techniques. Gap junctions were restricted to the proximal tubule, which is consistent with other vertebrate classes. Segments I and II of the proximal tubule and the collecting tubule/collecting duct system exhibited a well-developed zonula occludens with anastomosing strands. The distal segment showed a narrow zonula occludens composed of few parallel strands. The structure of the occluding junctions along the renal tubule of this teleost displays several similarities with the pattern of the zonulae occludentes in the amphibian and the mammalian nephron. From these observations, in conjunction with available data from other vertebrate classes, it can be concluded that in the proximal tubule the development of a deep and complex zonula occludens is a general feature of cold-blooded vertebrates.     

Intercellular junctions during development of the definitive kidney in lampreys: freeze fracture and morphometric analyses

The changing morphology of intercellular junctions in renal morphogenesis during lamprey metamorphosis was followed using freeze-fracture replicas and morphometry. Gap junctions and particle aggregates among strands of occluding junctions are conspicuous in the differentiating podocytes of the renal corpuscle and in the early ciliated neck and proximal segments but not in the distal segment. The cells of the segmented nephron arise from alpha nephrogenic cells which have a focal aggregate (macula occludens) of 4.8 junctional strands. Upon the initiation of metamorphosis the number of strands increases to 8 in undifferentiated cells with either maculae or zonulae occludentes. Differentiation of both neck and proximal segments is accompanied by gradual transformation of a 7-strand zonula occludens to a 4-strand junction but it becomes more shallow in the latter segment. Two types of undifferentiated cell are recognized through five of the seven metamorphic stages. Cells of two types of distal segments begin differentiation at the midpoint of transformation and immediately show zonula occludens of different morphology. Distal segment I (pars recta) has 5 strands and a 0.250 mum depth whereas segment II has 8-9 strands with twice the apical-basal depth. The larval archinephric duct undergoes a moderate transformation in junctional morphology with the addition of 2 strands and no increase in apical-basal depth in zonulae occludentes during metamorphosis. Changes and development of types of intercellular junctions along the nephron in lampreys are discussed with reference to known regional functional specialization in this organism and with the morphology of renal tubular intercellular junctions in other vertebrates.     

Gap junctions assemble in the presence of cytoskeletal inhibitors, but enhanced assembly requires microtubules

The role of cytoskeletal elements in gap junction (GJ) assembly has been studied using Novikoff hepatoma cells treated with cytochalasin B (CB) to disrupt actin filaments or with colchicine or nocodazole to disrupt microtubules. After 60 min of cell reaggregation, freeze-fracture was used to evaluate quantitatively the "initiation," "maturation," and "growth" phases of GJ assembly. The development of junctional permeability to fluorescent dyes was also analyzed. The only effects of CB on the structure or permeability of the developing junctions involved an elongation of GJ aggregates and a small decrease in formation plaque areas. Colchicine (but not the inactive form, lumicolchicine) prevented the enhancement of GJ growth by cholesterol, but its effect on basal growth was equivocal. Nocodazole inhibited the growth of GJ, even under basal conditions, without an effect on initiation. Nocodazole also blocked the forskolin-enhanced increase in the growth of GJs and, in living MDCK cells, reduced the movement of transport intermediates containing green fluorescent protein-tagged connexin43. Thus, neither actin filaments nor microtubules appear to restrict GJ assembly by anchoring intramembrane GJ proteins, nor are they absolutely required for functional GJs to form. However, microtubules are necessary for enhanced GJ growth and likely for facilitating connexin trafficking under basal conditions.     

Influence of metabolic inhibitors on the degradation of tight junctions in HT29 cells.

The human colon adenocarcinoma cell line HT 29 grows in culture without tight junctions (TJ). Tight junction strands of the fascia occludens type can be induced by treatment with proteases and are subsequently degraded during a period of about 3 h. Experiments using a variety of metabolic inhibitors such as 2-deoxyglucose, 2,4-dinitrophenol, and CCCP show that the degradation of TJ is retarded under conditions of ATP depletion. Thus it appears that the removal of TJ from the cell surface is an energy-dependent process. Moreover, DNP can specifically inhibit the degradation of TJ even in the absence of ATP depletion. The possible involvement of a proton gradient in the mechanism of TJ degradation is discussed.     

Distribution of Tight Junction Proteins in Adult Human Salivary Glands

Tight junctions (TJs) are an essential structure of fluid-secreting cells, such as those in salivary glands. Three major families of integral membrane proteins have been identified as components of the TJ: claudins, occludin, and junctional adhesion molecules (JAMs), plus the cytosolic protein zonula occludens (ZO). We have been working to develop an orally implantable artificial salivary gland that would be suitable for treating patients lacking salivary parenchymal tissue. To date, little is known about the distribution of TJ proteins in adult human salivary cells and thus what key molecular components might be desirable for the cellular component of an artificial salivary gland device. Therefore, the aim of this study was to determine the distribution of TJ proteins in human salivary glands. Salivary gland samples were obtained from 10 patients. Frozen and formalin-fixed paraffin-embedded sections were stained using IHC methods. Claudin-1 was expressed in ductal, endothelial, and ∼25% of serous cells. Claudins-2, -3, and -4 and JAM-A were expressed in both ductal and acinar cells, whereas claudin-5 was expressed only in endothelial cells. Occludin and ZO-1 were expressed in acinar, ductal, and endothelial cells. These results provide new information on TJ proteins in two major human salivary glands and should serve as a reference for future studies to assess the presence of appropriate TJ proteins in a tissue-engineered human salivary gland. (J Histochem Cytochem 56:1093–1098, 2008)     

Alcohol-induced blood-brain barrier dysfunction is mediated via inositol 1,4,5-triphosphate receptor (IP3R)-gated intracellular calcium release

The blood-brain barrier (BBB) formed by brain microvascular endothelial cells (BMVEC), pericytes and astrocytes controls the transport of ions, peptides and leukocytes in and out of the brain. Tight junctions (TJ) composed of TJ proteins (occludin, claudins and zonula occludens) ensure the structural integrity of the BMVEC monolayer. Neuropathologic studies indicated that the BBB was impaired in alcohol abusers; however, the underlying mechanism of BBB dysfunction remains elusive. Using primary human BMVEC, we previously demonstrated that oxidative stress induced by ethanol (EtOH) metabolism in BMVEC activated myosin light chain kinase (MLCK), resulting in the enhanced phosphorylation of either cytoskeletal or TJ proteins, and in BBB impairment. We proposed that EtOH metabolites stimulated inositol 1,4,5-triphosphate receptor (IP(3)R)-operated intracellular calcium (Ca(2+)) release, thereby causing the activation of MLCK in BMVEC. Indeed, treatment of primary human BMVEC with EtOH or its metabolites resulted in the increased expression of IP(3)R protein and IP(3)R-gated intracellular Ca(2+) release. These functional changes paralleled MLCK activation, phosphorylation of cytoskeletal/TJ proteins, loss of BBB integrity, and enhanced leukocyte migration across BMVEC monolayers. Inhibition of either EtOH metabolism or IP(3)R activation prevented BBB impairment. These findings suggest that EtOH metabolites act as signaling molecules for the activation of MLCK via the stimulation of IP(3)R-gated intracellular Ca(2+) release in BMVEC. These putative events can lead to BBB dysfunction in the setting of alcoholism, and to neuro-inflammatory disorders promoting leukocyte migration across the BBB.     

Remodeling the zonula adherens in response to tension and the role of afadin in this response

Morphogenesis requires dynamic coordination between cell–cell adhesion and the cytoskeleton to allow cells to change shape and move without losing tissue integrity. We used genetic tools and superresolution microscopy in a simple model epithelial cell line to define how the molecular architecture of cell–cell zonula adherens (ZA) is modified in response to elevated contractility, and how these cells maintain tissue integrity. We previously found that depleting zonula occludens 1 (ZO-1) family proteins in MDCK cells induces a highly organized contractile actomyosin array at the ZA. We find that ZO knockdown elevates contractility via a Shroom3/Rho-associated, coiled-coil containing protein kinase (ROCK) pathway. Our data suggest that each bicellular border is an independent contractile unit, with actin cables anchored end-on to cadherin complexes at tricellular junctions. Cells respond to elevated contractility by increasing junctional afadin. Although ZO/afadin knockdown did not prevent contractile array assembly, it dramatically altered cell shape and barrier function in response to elevated contractility. We propose that afadin acts as a robust protein scaffold that maintains ZA architecture at tricellular junctions.     

Development of cell-to-cell relationships in the thyroid gland of the chick embryo

Summary Thyroid glands of 7 to 21 day-old chick embryos were examined by electron microscopy using freeze-fracture and thin-section preparations. The primitive follicle lumen first appears between two adjacent epithelial cells in 8 day-old embryos, and is formed in the region of a focal tight junction (macula occludens). The focal tight junction develops into the zonula occludens when the primitive follicle lumen first forms.The zonula occludens is, at first, composed of 4.6 ± 2.45 strands, but with increasing embryonic age the number of strands increases to 5.9 ± 1.41 in 13 day-old, and 8.0 ± 1.75 in 19 day-old embryos.Thyroglobulin stored within the embryonic gland lumina is isolated from the mesenchymal tissue even at the first appearance of these follicle cavities.Well developed gap junctions already occur in the thyroid gland of the 7 day-old embryo, so that an intimate relationship and communication between these cells already exists at the time of their functional differentiation.This study was supported by a grant from the Japan Ministry of Education     

Microtubule Integrity Is Necessary for the Epithelial Barrier Function of Cultured Thyroid Cell Monolayers

Vectorial transport in the thyroid epithelium requires an efficient barrier against passive paracellular flux, a role which is principally performed by the tight junction (zonula occludens). There is increasing evidence that tight junction integrity is determined by integral and peripheral membrane proteins which interact with the cell cytoskeleton. Although the contribution of the actin cytoskeleton to tight junction physiology has been intensively studied, less is known about possible interactions with microtubules. In the present study we used electrophysiological and immunohistochemical approaches to investigate the contribution of microtubules to the paracellular barrier in cultured thyroid cell monolayers which displayed a high transepithelial electrical resistance (6000-9000 ohm · cm²). Colchicine (1 μM) caused a progressive fall in electrical resistance to <10% of baseline after 6 h and depolarization of the transepithelial electrical potential difference consistent with a significant increase in paracellular permeability. The effect of colchicine on TER was not affected by agents which inhibit the major apical conductances of thyroid cells but was reversed upon removal of the drug. Immunofluorescent staining for tubulin combined with confocal laser scanning microscopy demonstrated that thyroid cells possessed a dense microtubule network extending throughout the cytoplasm which was destroyed by colchicine. Colchicine also produced changes in the localization of the tight junction-associated protein, ZO-1: its normally continuous junctional distribution was disrupted by striking discontinuities and the appearance of many fine strands which extended into the cytoplasm. A similar disruption in E-cadherin staining was also observed, but colchicine did not affect the distribution of vinculin associated with adherens junctions nor the integrity of the perijunctional actin ring. We conclude that microtubules are necessary for the functional and structural integrity of tight junctions in this electrically tight, transporting epithelium.     

Zonula occludens-1 alters connexin43 gap junction size and organization by influencing channel accretion

Regulation of gap junction (GJ) organization is critical for proper function of excitable tissues such as heart and brain, yet mechanisms that govern the dynamic patterning of GJs remain poorly defined. Here, we show that zonula occludens (ZO)-1 localizes preferentially to the periphery of connexin43 (Cx43) GJ plaques. Blockade of the PDS95/dlg/ZO-1 (PDZ)-mediated interaction between ZO-1 and Cx43, by genetic tagging of Cx43 or by a membrane-permeable peptide inhibitor that contains the Cx43 PDZ-binding domain, led to a reduction of peripherally associated ZO-1 accompanied by a significant increase in plaque size. Biochemical data indicate that the size increase was due to unregulated accumulation of gap junctional channels from nonjunctional pools, rather than to increased protein expression or decreased turnover. Coexpression of native Cx43 fully rescued the aberrant tagged-connexin phenotype, but only if channels were composed predominately of untagged connexin. Confocal image analysis revealed that, subsequent to GJ nucleation, ZO-1 association with Cx43 GJs is independent of plaque size. We propose that ZO-1 controls the rate of Cx43 channel accretion at GJ peripheries, which, in conjunction with the rate of GJ turnover, regulates GJ size and distribution.     

Larazotide acetate regulates epithelial tight junctions in vitro and in vivo

Tight junctions (TJs) control paracellular permeability and apical-basolateral polarity of epithelial cells, and can be regulated by exogenous and endogenous stimuli. Dysregulated permeability is associated with pathological conditions, such as celiac disease and inflammatory bowel disease. Herein we studied the mechanism by which larazotide acetate, an 8-mer peptide and TJ regulator, inhibits the cellular changes elicited by gliadin fragments, AT-1002, and cytokines. Previously, we demonstrated that AT-1002, a 6-mer peptide derived from the Vibrio cholerae zonula occludens toxin ZOT, caused several biochemical changes in IEC6 and Caco-2 cells resulting in decreased transepithelial electrical resistance (TEER) and increased TJ permeability. In this study, larazotide acetate inhibited the redistribution and rearrangement of zonula occludens-1 (ZO-1) and actin caused by AT-1002 and gliadin fragments in Caco-2 and IEC6 cells. Functionally, larazotide acetate inhibited the AT-1002-induced TEER reduction and TJ opening in Caco-2 cells. Additionally, larazotide acetate inhibited the translocation of a gliadin 13-mer peptide, which has been implicated in celiac disease, across Caco-2 cell monolayers. Further, apically applied larazotide acetate inhibited the increase in TJ permeability elicited by basolaterally applied cytokines. Finally, when tested in vivo in gliadin-sensitized HLA-HCD4/DQ8 double transgenic mice, larazotide acetate inhibited gliadin-induced macrophage accumulation in the intestine and preserved normal TJ structure. Taken together, our data suggest that larazotide acetate inhibits changes elicited by AT-1002, gliadin, and cytokines in epithelial cells and preserves TJ structure and function in vitro and in vivo.