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PubMed2004

Hepatocyte growth factor disrupts tight junctions in human breast cancer cells.

Tight junctions in epithelial cells act as cell-cell adhesion structures and govern paracellular permeability. Disruption of these functions can lead to dissociation of cancer cells. This study aimed to determine whether HGF, a cytokine secreted by stromal cells, is capable of modulating expression and function of tight junction molecules in human breast cancer cell lines. HGF decreased trans-epithelial resistance and increased paracellular permeability of two human breast cancer cell lines, MDA MB 231 and MCF-7. Q-PCR showed that HGF modulated the levels of several tight junction molecule (occludin, claudin-1 and -5, JAM-1 and -2) mRNA transcripts in MDA MB 231 and MCF-7 cells. Western blotting and immunohistochemistry also showed modulation of expression of the tight junction molecule, occludin. It is suggested that HGF disrupts tight junction function in human breast cancer cells by effecting changes in the expression of tight junction molecules at both the mRNA and protein levels. We conclude that regulation of tight junctions could be of fundamental importance in the prevention of metastasis of breast cancer cells.

Breast Neoplasms↗
PubMed1989

The gastric mucosal barrier: tight junction structure in gastritis and ulcer biopsies.

Tight junctions of the human gastric mucosa were examined using quantitative freeze-fracture methods. Biopsies examined were from patients with gastric diseases including gastritis, ulcers, and pernicious anemia. No significant differences were seen in strand number or tight junction complex depth among the biopsies analyzed, however, anomalous tight junction structures were observed. Discontinuities in the tight junctions complex and hyperplastic tight junctions (extensions of the apical tight junction strands radiating over the lateral plasma membrane) were seen. These alterations were not associated exclusively with either the diagnosis of gastritis or ulcers. However, a higher frequency of tight junction breaks was seen in stomach biopsies diagnosed as gastritis while those diagnosed as ulcers displayed a higher occurrence of hyperplastic tight junctions.

Adult↗
PubMed2001

Cx32 but not Cx26 is associated with tight junctions in primary cultures of rat hepatocytes.

On freeze-fracture replicas, gap junctions are frequently colocalized with tight junctions. In this study, to elucidate the relationship between gap- and tight-junction proteins, we investigated the localization of gap-junction proteins Cx32 and Cx26 and tight-junction proteins occludin, claudin-1, ZO-1, and ZO-2 in primary cultured rat hepatocytes, using confocal laser microscopy. In hepatocytes cultured in 2% DMSO and 10(-7) M glucagon medium, Cx32- but not Cx26-immunoreactive lines were observed on the most subapical plasma membrane at cell borders, while on the basolateral membrane both Cx32- and Cx26-positive spots were colocalized. Occludin-, claudin-1-, ZO-1-, and ZO-2-immunoreactive lines were also linearly observed on the most subapical plasma membrane and were colocalized with only Cx32-immunoreactive lines. In freeze-fracture analysis, many small gap-junction plaques were observed within a well-developed tight-junction strand network. The fence function of tight junctions in the cells, as examined by diffusion of labeled sphingomyelin, was well maintained. We also carried out Western blotting for Cx32 following immunoprecipitation with anti-occludin, anti-claudin-1, or anti-ZO-1 antibodies. Cx32 was detectable in all immunoprecipitates. These results suggest that Cx32 gap junctions, but not those with Cx26, are closely coordinated with the expression and function of tight junctions in hepatocytes and that Cx32 gap-junction formation may affect cell polarity through modification of tight-junction expression.

Animals↗
PubMed2006

Tight junctions and cell polarity.

The tight junction is an intracellular junctional structure that mediates adhesion between epithelial cells and is required for epithelial cell function. Tight junctions control paracellular permeability across epithelial cell sheets and also serve as a barrier to intramembrane diffusion of components between a cell's apical and basolateral membrane domains. Recent genetic and biochemical studies in invertebrates and vertebrates indicate that tight junction proteins play an important role in the establishment and maintenance of apico-basal polarity. Proteins involved in epithelial cell polarization form evolutionarily conserved multiprotein complexes at the tight junction, and these protein complexes regulate the architecture of epithelia throughout the polarization process. Accumulating information regarding the regulation of these polarity proteins will lead to a better understanding of the molecular mechanisms whereby cell polarity is established.

Animals↗
PubMed1999

Manner of interaction of heterogeneous claudin species within and between tight junction strands.

In tight junctions (TJs), TJ strands are associated laterally with those of adjacent cells to form paired strands to eliminate the extracellular space. Claudin-1 and -2, integral membrane proteins of TJs, reconstitute paired TJ strands when transfected into L fibroblasts. Claudins comprise a multigene family and more than two distinct claudins are coexpressed in single cells, raising the questions of whether heterogeneous claudins form heteromeric TJ strands and whether claudins interact between each of the paired strands in a heterophilic manner. To answer these questions, we cotransfected two of claudin-1, -2, and -3 into L cells, and detected their coconcentration at cell-cell borders as elaborate networks. Immunoreplica EM confirmed that distinct claudins were coincorporated into individual TJ strands. Next, two L transfectants singly expressing claudin-1, -2, or -3 were cocultured and we found that claudin-3 strands laterally associated with claudin-1 and -2 strands to form paired strands, whereas claudin-1 strands did not interact with claudin-2 strands. We concluded that distinct species of claudins can interact within and between TJ strands, except in some combinations. This mode of assembly of claudins could increase the diversity of the structure and functions of TJ strands.

Animals↗
PubMed2003

Claudin extracellular domains determine paracellular charge selectivity and resistance but not tight junction fibril architecture.

Tight junctions (TJs) regulate paracellular permeability across epithelia and vary widely in their transepithelial electrical resistance (TER) and charge selectivity. The claudin family of transmembrane proteins influences these properties. We previously reported that claudin-4 increased TER approximately 300% when expressed in low-resistance Madin-Darby canine kidney (MDCK) II cells and decreased the paracellular permeability for Na(+) more than Cl(-) (Van Itallie C, Rahner C, and Anderson JM. J Clin Invest 107: 1319-1327, 2001). In comparison, we report here that expression of claudin-2 increases TER by only approximately 20% and does not change the ionic selectivity of MDCK II cells from their cation-selective background. To test whether the extracellular domains of claudins-4 and -2 determine their unique paracellular properties, we determined the effects of interchanging these domains between claudins-4 and -2. Inducible expression of wild-type claudins and extracellular domain chimeras increased both the number and depth of fibrils, but the characteristic fibril morphologies of claudin-4 or -2 were not altered by switching extracellular domains. Like claudin-4, chimeras expressing the first or both extracellular domains of claudin-4 on claudin-2 increased TER severalfold and profoundly decreased the permeability of Na(+) relative to Cl(-). In contrast, chimeras expressing the first or both extracellular domains of claudin-2 on claudin-4 increased the TER by only approximately 60 and approximately 40%, respectively, and only modestly altered charge selectivity. These results support a model in which the claudins create paracellular channels and the first extracellular domain is sufficient to determine both paracellular charge selectivity and TER.

Amino Acid Sequence↗
PubMed1998

Testin secreted by Sertoli cells is associated with the cell surface, and its expression correlates with the disruption of Sertoli-germ cell junctions but not the inter-Sertoli tight junction.

Testin is a testosterone-responsive Sertoli cell secretory product. In the present study, we demonstrated that the amount of testin secreted by Sertoli cells in vitro was comparable with several other Sertoli cell secretory products. However, virtually no testin was found in the luminal fluid and cytosols of the testis and epididymis when the intercellular junctions were not previously disrupted, suggesting that secreted testin may be reabsorbed by testicular cells in vivo. Studies using Sertoli cells with and without a cell surface cross-linker and radioiodination in conjunction with immunoprecipitation illustrated the presence of two polypeptides of 28 and 45 kDa, which constitute a binding protein complex that anchors testin onto the cell surface. The 28- and 45-kDa peptide appear to be residing on and inside the cell surface, respectively. Immunogold EM studies illustrated testin was abundantly localized on the Sertoli cell side of the ectoplasmic specialization (a modified adherens junction) surrounding developing spermatids. In contrast, very few testin gold particles were found at the site of inter-Sertoli tight junctions. When the inter-Sertoli tight junctions were formed or disrupted, no significant change in testin expression was noted. This is in sharp contrast to the disruption of Sertoli-germ cell junctions, which is accompanied by a surge in testin expression. These results demonstrate the usefulness of testin in examining Sertoli-germ cell interactions.

Aging↗
PubMed2002

Role of tight junction derangement in the endothelial dysfunction elicited by exogenous and endogenous peroxynitrite and poly(ADP-ribose) synthetase.

DNA single-strand breakage and activation of the nuclear enzyme poly(ADP-ribose) synthetase (PARS) triggers an energy consuming, inefficient repair cycle, which contributes to peroxynitrite-induced cellular injury. Here, we investigated whether peroxynitrite and PARS activation are involved in tight junctions (tight junction) derangement in the endothelial dysfunction in cells exposed to peroxynitrite and in vascular rings of animals subjected to zymosan non-septic shock. In human umbilical vein endothelial cells (HUVEC) in vitro, peroxynitrite caused a dose-dependent suppression of mitochondrial respiration, as measured by the mitochondrial-dependent conversion of the dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide to formazan. Moreover, peroxynitrite caused activation of PARS. Inhibition of PARS by 3-aminobenzamide (3-AB; 1 mM) reduced the peroxynitrite-induced suppression of mitochondrial respiration in HUVECs. Vascular rings exposed to peroxynitrite exhibited reduced endothelium-dependent relaxant responses in response to acetylcholine. Peroxynitrite incubation also caused a significant derangement of zonula occludens (ZO)-1, which was significantly affected by pharmacological inhibition of PARS. 3-AB ameliorated the development of this peroxynitrite-induced endothelial dysfunction. In vascular rings obtained from the zymosan-treated rats, there was a marked suppression of the endothelium-dependent relaxation ex vivo, which was reduced by in vivo 3-AB treatment. A significant derangement of ZO-1 was observed in vascular rings from zymosan-treated rats. Tight junction alteration was significantly reduced by in vivo 3-AB treatment. Thus, activation of PARS by exogenous and endogenous peroxynitrite may be involved in the tight junction derangement associated with endothelial dysfunction. Inhibition of PARS may be a novel pharmacological approach to preserve endothelial tight junction function in shock and inflammation.

Actins↗
PubMed1998

The tight junction: morphology to molecules.

The tight junction forms a regulated barrier in the paracellular pathway between epithelial and endothelial cells. This intercellular junction also demarcates the compositionally distinct apical and basolateral membranes. While the existence of a paracellular barrier in epithelia was hypothesized by physiologists over a century ago, the molecular characterization of the tight junction is a relatively new and rapidly expanding area of research. It is now recognized that the tight junction is comprised of at least nine peripheral and one integral membrane proteins. This complex includes members of a protein family related to tumor suppression and signal transduction, a rab protein, and a Ras target protein. The characteristics of, interactions between, and potential physiological roles of these proteins at the tight junction are discussed.

Animals↗
PubMed2005

Tight junctions and bladder cancer (review).

Tight junctions play a critical role in the maintenance of the urine-blood barrier creating a physiological barrier to the passage of ions and solutes between the urine and blood. Alterations in this urine-blood barrier function have been demonstrated in some diseases and regulation of the tight junction function has been recognised as an important aspect of the cell biology of cancer in terms of disease progression and as a potential therapeutic target. Although tight junctions play an important role in the physiological control of bladder function, there is little published on their molecular composition or regulation in the normal or diseased bladder. The purpose of this review is to summarise current understanding on the role and regulation of tight junction function in the normal and diseased bladder.

Cell Movement↗
PubMed2004

Identification of a novel protein, LYRIC, localized to tight junctions of polarized epithelial cells.

Tight junctions (TJ) are multiprotein complexes that function to regulate paracellular transport of molecules through epithelial and endothelial cell layers. Many new tight junction-associated proteins have been identified in the past few years, and their functional roles and interactions have just begun to be elucidated. In this paper, we describe a novel protein LYsine-RIch CEACAM1 co-isolated (LYRIC) that is widely expressed and highly conserved between species. LYRIC has no conserved domains that would indicate function and does not appear to be a member of a larger protein family. Data from analysis of rat and human tissue sections and cell lines show that LYRIC colocalizes with tight junction proteins ZO-1 and occludin in polarized epithelial cells, suggesting that LYRIC is part of the tight junction complex. LYRIC dissociates from ZO-1 when junctional complexes are disrupted, and as tight junctions reform, ZO-1 relocalizes before LYRIC. These results suggest that LYRIC is most likely not a structural component required for TJ formation, but rather is recruited during the maturation of the tight junction complex.

Animals↗
PubMed1991

Hydrophobic ion transfer between membranes of adjacent hepatocytes: a possible probe of tight junction structure.

The topology of the tight junction is probed by introducing dipicrylamine (dpa-), a lipid-soluble anion, into the membranes of hepatocyte pairs in culture. Once partitioned into the membrane, dpa- ions are free to move in the hydrophobic core of the membrane, where their mobile charges greatly increase membrane capacitance. If tight junctions are lines of membrane fusion, dpa- will cross the tight junction without traversing a polar headgroup layer. Furthermore, the electric potential across the tight junction will be equal to the difference in membrane potentials of the two cells. dpa- can therefore be expected to move electrophoretically from cell membrane to cell membrane across the junction in response to an intercellular voltage difference. Experiments performed under double whole-cell clamp show that this transfer occurs as follows: First, dpa- causes an intercellular current unrelated to gap junctions to flow in response to an intercellular voltage difference. Second, this electrophoretic removal or addition of dpa- from a cell's membrane through the tight junction must reduce or increase its dpa- content and thus its capacitance. Experiments confirm this prediction: We detect rapid, symmetric, and reversible changes in membrane capacitance in response to changes in the membrane potential of the neighboring cell. Finally, we find that hepatocyte membranes contain a negatively charged endogenous molecule that contain a negatively charged endogenous molecule that can move from cell to cell like dpa- under the influence of an intercellular potential difference. We conclude that membrane fusion occurs at tight junctions and that this hydrophobic intercellular pathway can play a role in intercellular communication.

Animals↗
PubMed2005

Production of ammonium by Helicobacter pylori mediates occludin processing and disruption of tight junctions in Caco-2 cells.

Tight junctions, paracellular permeability barriers that define epithelial cell polarity, play an essential role in transepithelial transport, cell-cell adhesion and lymphocyte transmigration. They are also important for the maintenance of innate immune defence and intestinal antigen uptake. Ammonium (NH4+) is elevated in the gastric aspirates of Helicobacter pylori-infected patients and has been implicated in the disruption of tight-junction functional integrity and the induction of gastric mucosal damage during H. pylori infection. The precise mechanism of the effect of ammonium and the molecular targets of ammonium in host tissue are not yet identified. To study the effects of ammonium on epithelial tight junctions, the human colon carcinoma cell line Caco-2 was cultured on permeable supports and the transepithelial resistance (TER) was measured at different time intervals following exposure to ammonium salts or H. pylori-derived ammonium. A biphasic response to treatment with ammonium was found. Acute exposure to ammonium salts or NH3/NH4+ derived from urea metabolism by wild-type H. pylori resulted in a 20-30 % decrease in TER. After 24 h, the NH4Cl-treated cells showed a partial recovery of TER. In contrast, the control culture, or cultures that were exposed to supernatants derived from urease-deficient H. pylori, showed no significant decrease in TER. Occludin-specific immunoblots revealed the expression of a low-molecular-weight form of occludin of 42 kDa upon NH3/NH4+ exposure. The results indicate that modulation of tight-junction function by H. pylori is ammonium-dependent and linked to the accumulation of a low-molecular-weight and detergent-soluble form of occludin.

Caco-2 Cells↗
PubMed2002

Cx32 formation and/or Cx32-mediated intercellular communication induces expression and function of tight junctions in hepatocytic cell line.

Gap junctional intercellular communication (GJIC) is thought to play a crucial role in cell differentiation. Small gap junction plaques are frequently associated with tight junction strands in hepatocytes, suggesting that gap junctions may be closely related to the role of tight junctions in the establishment of cell polarity. To examine the exact role of gap junctions in regulating tight junctions, we transfected connexin 32 (Cx32), Cx26, or Cx43 cDNAs into immortalized mouse hepatocytes derived from Cx32-deficient mice and examined the expression and function of the endogenous tight junction molecules. In transient wild-type Cx32 transfectants, immunocytochemistry revealed that endogenous occludin was in part localized at cell borders, where it was colocalized with Cx32, whereas neither was detected in parental cells. In Cx32 null hepatocytes transfected with Cx32 truncated at position 220 (R220stop), wild-type Cx26, or wild-type Cx43 cDNAs, occludin was not detected at cell borders. In stable wild-type Cx32 transfectants, occludin, claudin-1, and ZO-1 mRNAs and proteins were significantly increased compared to parental cells and all of the proteins were colocalized with Cx32 at cell borders. Treatment with a GJIC blocker, 18 beta-glycyrrhetinic acid, resulted in decreases of occludin and claudin-1 at cell borders in the stable transfectants. The induction of tight junction proteins in the stable transfectants was accompanied by an increase in both fence and barrier functions of tight junctions. Furthermore, in the stable transfectants, circumferencial actin filaments were also increased without a change of actin protein. These results indicate that Cx32 formation and/or Cx32-mediated intercellular communication may participate in the formation of functional tight junctions and actin organization.

Actin Cytoskeleton↗
PubMed1980

Definitive evidence for the existence of tight junctions in invertebrates.

Extensive and unequivocal tight junctions are here reported between the lateral borders of the cellular layer that circumscribes the arachnid (spider) central nervous system. This account details the features of these structures, which form a beltlike reticulum that is more complex than the simple linear tight junctions hitherto found in invertebrate tissues and which bear many of the characteristics of vertebrate zonulae occludentes. We also provide evidence that these junctions form the basis of a permeability barrier to exogenous compounds. In thin sections, the tight junctions are identifiable as punctate points of membrane apposition; they are seen to exclude the stain and appear as election- lucent moniliform strands along the lines of membrane fusion in en face views of uranyl-calcium-treated tissues. In freeze-fracture replicas, the regions of close membrane apposition exhibit P-face (PF) ridges and complementary E-face (EF) furrows that are coincident across face transitions, although slightly offset with respect to one another. The free inward diffusion of both ionic and colloidal lanthanum is inhibited by these punctate tight junctions so that they appear to form the basis of a circumferential blood-brain barrier. These results support the contention that tight junctions exist in the tissues of the invertebrata in spite of earlier suggestions that (a) they are unique to vertebrates and (b) septate junctions are the equivalent invertebrate occluding structure. The component tight junctional 8- to 10-nm-particulate PF ridges are intimately intercalated with, but clearly distinct from, inverted gap junctions possessing the 13-nm EF particles typical of arthropods. Hence, no confusion can occur as to which particles belong to each of the two junctional types, as commonly happens with vertebrate tissues, especially in the analysis of developing junctions. Indeed, their coexistance in this way supports the idea, over which there has been some controversy, that the intramembrane particles making up these two junctional types must be quite distinct entities rather than products of a common precursor.

Animals↗
PubMed2005

Multiple regions of Crumbs3 are required for tight junction formation in MCF10A cells.

The formation and maintenance of tight junctions is essential for the development of epithelial cell polarity. Recently, a number of conserved polarity-regulating proteins have been shown to localize to epithelial tight junctions, and to play a role in the regulation of tight junction formation. The Crumbs3/PALS1/PATJ protein complex localizes at epithelial tight junctions and interacts with the polarity-regulating protein complex of Par6/Par3/aPKC. Overexpression of Crumbs3 in MDCKII cells leads to a delay in tight junction formation in these cells, suggesting a role in the regulation of tight junction development. Here we report new evidence that Crumbs3 indeed plays an essential role in tight junction formation. Mammary MCF10A cells express little endogenous Crumbs3 and fail to form tight junctions when grown under standard tissue culture conditions. The staining pattern of ZO-1, a tight junction marker, is fragmented, and other tight junction markers show either fragmented junctional expression or diffuse cytoplasmic staining. Expression of exogenous Crumbs3 induces the formation of tight junction structures marked by smooth, continuous ZO-1 staining at apical cell-cell junctions. A number of other tight junction markers, including claudin-1 and occludin, are also recruited to these junctions. Analysis by transmission electron microscopy and measurements of the transepithelial electrical resistance confirm that these structures are functional tight junctions. Mutations in either the Crumbs3 PDZ binding motif or the putative FERM binding motif lead to defects in the ability of Crumbs3 to promote tight junction development. Our results suggest that Crumbs3 plays an important role in epithelial tight junction formation, and also provide the first known functional role for the mammalian Crumbs FERM binding domain.

Amino Acid Sequence↗
PubMed1989

Development of tight junctions de novo in the mouse early embryo: control of assembly of the tight junction-specific protein, ZO-1.

Tight junction development during trophectoderm biogenesis in the mouse preimplantation embryo has been examined using monoclonal antibodies recognizing the tight junction-specific peripheral membrane protein, ZO-1. In immunoblots, mouse embryo ZO-1 had a molecular mass (225 kD) equivalent to that in mouse liver, was barely detectable in four-cell embryos although later stages exhibited increasing levels. ZO-1 was first detected immunocytochemically at the compacting eight-cell stage, coincident with or just after the expression of basolateral cell adhesion and apical microvillous polarity. Initially, ZO-1 was present as a series of spots along the boundary between free and apposed cell surfaces in intact embryos or cell couplets, but subsequently staining became more linear with blastocyst trophectoderm cells being bordered by a continuous ZO-1 belt. Inhibition of cell adhesion at the 8-cell stage delayed ZO-1 appearance and randomized its surface distribution in a reversible manner. Microfilament disruption, but not microtubule depolymerization, produced major disturbances in ZO-1 distribution. ZO-1 assembly de novo appeared to be independent of proximate DNA and RNA synthesis but was inhibited substantially in the absence of protein synthesis during the eight-cell stage, a treatment that did not prevent intercellular adhesion and polarization. ZO-1 surface assembly, but not adhesion and polarization, was also perturbed when single eight-cells were combined with single four-cells. The results suggest that tight junction development in mouse embryos is a secondary event in epithelial biogenesis, being dependent upon cell adhesion and cytoskeletal activity for normal expression, and can be disrupted without disturbing the generation of a stably polarized phenotype.

Animals↗
PubMed1997

Physiological regulation of epithelial tight junctions is associated with myosin light-chain phosphorylation.

Tight junctions serve as the rate-limiting barrier to passive movement of hydrophilic solutes across intestinal epithelia. After activation of Na+-glucose cotransport, the permeability of intestinal tight junctions is increased. Because previous analyses of this physiological tight junction regulation have been restricted to intact mucosae, dissection of the mechanisms underlying this process has been limited. To characterize this process, we have developed a reductionist model consisting of Caco-2 intestinal epithelial cells transfected with the intestinal Na+-glucose cotransporter, SGLT1. Monolayers of SGLT1 transfectants demonstrate physiological Na+-glucose cotransport. Activation of SGLT1 results in a 22 +/- 5% fall in transepithelial resistance (TER) (P < 0.001). Similarly, inactivation of SGLT1 by addition of phloridzin increases TER by 24 +/- 2% (P < 0.001). The increased tight junction permeability is size selective, with increased flux of small nutrient-sized molecules, e.g., mannitol, but not of larger molecules, e.g., inulin. SGLT1-dependent increases in tight junction permeability are inhibited by myosin light-chain kinase inhibitors (20 microM ML-7 or 40 microM ML-9), suggesting that myosin regulatory light-chain (MLC) phosphorylation is involved in tight junction regulation. Analysis of MLC phosphorylation showed a 2.08-fold increase after activation of SGLT1 (P < 0.01), which was inhibited by ML-9 (P < 0.01). Thus monolayers incubated with glucose and myosin light-chain kinase inhibitors are comparable to monolayers incubated with phloridzin. ML-9 also inhibits SGLT1-mediated tight junction regulation in small intestinal mucosa (P < 0.01). These data demonstrate that epithelial cells are the mediators of physiological tight junction regulation subsequent to SGLT1 activation. The intimate relationship between tight junction regulation and MLC phosphorylation suggests that a critical step in regulation of epithelial tight junction permeability may be myosin ATPase-mediated contraction of the perijunctional actomyosin ring and subsequent physical tension on the tight junction.

Animals↗