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PubMed2003

Tight junction proteins claudin-3 and claudin-4 are frequently overexpressed in ovarian cancer but not in ovarian cystadenomas.

PURPOSE: Claudin proteins represent a large family of integral membrane proteins crucial for tight junction (TJ) formation and function. Claudins have been shown to be up-regulated in various cancers and have been suggested as possible biomarkers and targets for cancer therapy. Because claudin-3 and claudin-4 have been proposed to be expressed in epithelial ovarian cancer, we have performed a detailed analysis of CLDN3 and CLDN4 expression in a panel of ovarian tumors of various subtypes and cell lines. We also investigated whether high expression of claudin-3 and claudin-4 was associated with TJ function in ovarian cancer cells. EXPERIMENTAL DESIGN: RNA was obtained from a panel of 39 microdissected epithelial ovarian tumors of various histological subtypes for real-time reverse transcription-PCR analysis. In addition, a total of 70 cases of ovarian carcinomas, ovarian cysts, and normal ovarian epithelium from a tissue array were analyzed by immunohistochemistry. Finally, a panel of cell lines was used for Western analysis of claudin expression and TJ permeability studies. RESULTS: Although expressed at low levels in some normal human tissues, including the ovary, CLDN3 and CLDN4 are highly up-regulated in epithelial ovarian cancers of all subtypes. Immunohistochemical analyses using our ovarian tissue array confirmed the high level of expression of claudin-3 and claudin-4 in the majority of ovarian carcinomas, including many tumors exhibiting cytoplasmic staining. Ovarian cystadenoma did not frequently overexpress these proteins, suggesting that the expression of these proteins is associated with malignancy. In ovarian cancer cell lines, claudin-3 and claudin-4 expression was not associated with functional TJs as measured by transepithelial electrical resistance. CONCLUSIONS: These results show that CLDN3 and CLDN4 are frequently up-regulated in ovarian tumors and cell lines and may represent novel markers for this disease. Overexpression of these genes in ovarian cancer also suggests interesting scenarios for the involvement of TJ in tumorigenesis. A better knowledge of the mechanisms underlying ovarian tumorigenesis will likely result in the development of novel approaches for the diagnosis and therapy of this deadly disease.

Blotting, Northern↗
PubMed1989

Establishment of tight junctions between cells from different animal species and different sealing capacities.

Epithelial cells establish tight junctions (TJs) that offer an ample range of transepithelial electrical resistances (TER), in adjustment to physiological requirements. In the present work, we demonstrate that cells from different animal origins, co-cultured in monolayers, can make sealed TJs, suggesting that this structure has a basic universal structure. TJs cannot be established, however, if one of the partners does not normally express TJs, indicating that each neighbor has to contribute its moiety. Furthermore, we observe that clones of the same cell line, with widely different values of TER, do not differ in the number and length of their junctional strands, suggesting that the difference is due to their ability to express ionic channels traversing their strands. The value of TER achieved in mixed monolayers of cells of the same or different lines is the one that may be expected by taking into account the proportion of each type in the mixture and adding in parallel the electrical resistance that they exhibit in pure monolayers. Therefore, epithelial TJs appear to behave as parallel resistances.

Animals↗
PubMed1991

Inhibition of adenine nucleotide synthesis: effect on tight junction structure and function of clone 4 MDCK cells.

The formation and maintenance of tight junctions as a barrier to the diffusion of ions and other water-soluble across epithelia is an energy-dependent process. The administration of N-formyl-hydroxyaminoacetic acid (Hadacidin), an analog of aspartate and a competitive inhibitor of adenylosuccinate synthetase, has been shown to inhibit the multiplication of clone 4 MDCK cells and concomitantly reduce the levels of ATP and cAMP (J. Cell. Physiol. 140, 186-194 (1989)). When added to mitotically quiescent confluent cultures of clone 4 MDCK cells, millimolar concentrations of Hadacidin inhibited the generation of transepithelial electrical resistance (TER). In such cultures passive Na+ permeability was similar to controls indicating that the effect of Hadacidin was not on the transcellular pathway. That these cells were viable was demonstrated by their ability to exclude Trypan Blue, and the fact that they remained competent to develop steady state TER upon removal of the inhibitor. Suppression of TER was completely reversed within 48 h of replacing the Hadacidin-supplemented medium with one containing aspartate. Adenosine, but not aspartate, when added simultaneously with the drug, obviated the latter's effect on TER. A mixture of dibutyryl cAMP (db-cAMP) and theophylline was only partially effective in overcoming the effects of Hadacidin on the development of TER and, in fact, markedly delayed its development in control cultures not treated with the drug. When monolayers with established steady state TER were exposed to Hadacidin, no change was noted during the first 24 h. By 48 h, however, TER had decreased to very low values.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenine Nucleotides↗
PubMed1993

Depleting cell cholesterol alters calcium-induced assembly of tight junctions by monolayers of MDCK cells.

A role for lipids in the formation of tight junctions (TJ) has been proposed. Attempts to relate changes in whole cell phospholipid composition to the formation of TJs, however, have yielded equivocal results. The object in the present study was to relate changes in TJ of MDCK cells more specifically to alterations in plasma membrane lipids. Cholesterol, which resides primarily in the plasma membrane, was reduced by 25% after incubation of cell monolayers for 24 h in a low Ca2+ medium supplemented with (1-2 microM) Lovastatin, an inhibitor of hydroxymethylglutarylcoenzyme A (HMG-CoA) reductase. This was associated with a halving of the time required for Ca2+ to induce TJ formation as monitored by transepithelial electrical resistance (TER). [3H]Mannitol flux, and morphometric measurements made on freeze fracture replicas confirm that the effects on TER reflect changes in the characteristics of the paracellular pathway. Peak and steady state values of TER were also elevated over control values. The changes in cholesterol content and the time course for TJ assembly were apparent at levels of Lovastatin which do not affect prenylation of proteins, and were prevented if 5 mM mevalonate was present along with Lovastatin. Paradoxically, despite a decrease of approximately 1/3 in the Ca concentration required to yield maximum rates of TJ assembly, 45Ca2+ uptake was actually reduced after cholesterol depletion. The data suggest that cholesterol may modulate the properties of membrane proteins and/or phospholipids which interact with Ca2+, possibly on the exoplasmic leaflet, during TJ assembly.

Animals↗
PubMed1999

Increased tight junctional permeability is associated with the development of colon cancer.

Epithelial tissues act as barriers between two fluid compartments, and the epithelial barrier function is provided by the epithelial cells and the tight junctions (TJs) that connect them. We have shown previously that chronic treatment of a cultured epithelial monolayer with phorbol ester tumor promoters induces an increase in transepithelial paracellular permeability and produces tumor-like polyps, suggesting an association between TJ permeability and tumor formation. In this study, we analyzed the association between TJ permeability and formation of tumors in vivo. The permeability of the TJs was assessed in normal human and rat colon epithelia and in colon tumors by measuring the transepithelial electrical resistance, the paracellular flux rate of D-[(14)C]mannitol and the electron microscopic evaluation of the penetration of the electron dense dye ruthenium red across the TJs. By these criteria, the TJs of human colon tumors, including carcinomas and adenomatous polyps, and the TJs of 1,2-dimethylhydrazine (DMH)-induced rat colon tumors were leakier than the TJs of normal colon. Treatment of rats with the carcinogen DMH induced a progressive increase in the number of aberrant colonic crypts, considered the putative pre-neoplastic colonic phenotype while increasing TJ permeability of the colon epithelium prior to the development of tumors. These results showed that increased TJ permeability of the colon epithelium and consequently a decrease in epithelial barrier function precede the development of colon tumors.

1,2-Dimethylhydrazine↗
PubMed2000

Cholestasis with altered structure and function of hepatocyte tight junction and decreased expression of canalicular multispecific organic anion transporter in a rat model of colitis.

Cholestasis is frequently associated with inflammatory bowel disease. Because some cholestasis is resulted from altered hepatocyte tight junctions (TJs) or the canalicular multispecific organic anion transporter, we have investigated the following topics in a rat model of inflammatory bowel disease: (1) alterations in hepatocyte TJs and in the canalicular multispecific organic anion transporter, (2) etiologic factors for cholestasis, and (3) effects of antibiotics on cholestasis. Rats with trinitrobenzene sulfonic acid-induced colitis were studied 24 hours after treatment. Hepatocyte TJs and the canalicular multispecific organic anion transporter were evaluated by immunostaining for TJ-associated proteins, 7H6 and ZO-1, and multidrug resistance protein 2 (mrp2). To investigate etiologic factors causing cholestasis, portal endotoxin and proinflammatory cytokines were examined. The effects of polymyxin B, penicillin G, or metronidazole on immunostaining for 7H6, ZO-1, mrp2, and cholestasis were investigated. (1) Immunostaining for 7H6 and ZO-1 colocalized outlining the bile canaliculi and immunostaining for mrp2 localized on the canalicular membrane in controls. Treatment with trinitrobenzene sulfonic acid induced significant cholestasis and caused translocation of immunostaining for 7H6, but not that for ZO-1, to the cytoplasm and diminished immunostaining for mrp2 on the canaliculus membrane. (2) The levels of portal endotoxin, but not proinflammatory cytokines, was increased. (3) Polymyxin B, but not the other antibiotics, prevented alterations in immunostaining for both 7H6 and mrp2, and cholestasis. We described that both hepatocyte TJs and the canalicular multispecific organic anion transporter were altered and that gut-derived endotoxin levels in the portal blood were increased in this rat colitis model.

Animals↗
PubMed2001

Perturbation of the tight junction permeability barrier by occludin loop peptides activates beta-catenin/TCF/LEF-mediated transcription.

Here we show that interference with the integrity of the transepithelial permeability barrier of mouse mammary epithelial cells by treatment with synthetic peptides, homologous to the second extracellular domain of occludin, decreased the amount of occludin protein present at tight junctions and led to the formation of multilayered, unpolarized cell clusters. In addition, transcription of the adherens junction protein beta-catenin was induced. Following accumulation of soluble beta-catenin protein, transcription by beta-catenin/TCF/LEF was increased, as revealed by transcriptional assays following transient transfection of the reporter construct. Furthermore, treatment with occludin-II peptides up-regulated RNA levels of the known beta-catenin/TCF/LEF downstream target gene c-myc. The data presented imply a functional cross-talk between tight and adherens junctions that possibly contributes to the stepwise transformation during oncogenesis.

Animals↗
PubMed2004

Tight junctions in differentiating ameloblasts and odontoblasts differentially express ZO-1, occludin, and claudin-1 in early odontogenesis of rat molars.

Little is known about the expression of associated proteins during the assembly of tight junctions (TJs). We studied the distribution of ZO-1, occludin, and claudin-1 between differentiating ameloblasts and odontoblasts in molar tooth germs from 1- to 3-day-old rats by confocal laser scanning microscopy. Immunoreactivity for ZO-1 was strong at proximal and distal junctional complexes of differentiating ameloblasts, while it was weak and punctuate at the distal region of differentiating odontoblasts. Occludin was immunoreactive at distal and proximal complexes of early differentiating ameloblasts and at distal regions of differentiating odontoblasts. However, in more advanced stages, occludin was only evident at the proximal complex of ameloblasts. Claudin-1 was strongly detected at the proximal complex but it was weak at distal complex of late differentiating ameloblasts. Thus, our results showed that ZO-1, occludin, and claudin-1 are differentially expressed as TJs assemble for regulating polarity and/or paracellular permeability in differentiating ameloblasts and odontoblasts.

Ameloblasts↗
PubMed2005

The Rac activator Tiam1 controls tight junction biogenesis in keratinocytes through binding to and activation of the Par polarity complex.

The GTPases Rac and Cdc42 play a pivotal role in the establishment of cell polarity by stimulating biogenesis of tight junctions (TJs). In this study, we show that the Rac-specific guanine nucleotide exchange factor Tiam1 (T-lymphoma invasion and metastasis) controls the cell polarity of epidermal keratinocytes. Similar to wild-type (WT) keratinocytes, Tiam1-deficient cells establish primordial E-cadherin-based adhesions, but subsequent junction maturation and membrane sealing are severely impaired. Tiam1 and V12Rac1 can rescue the TJ maturation defect in Tiam1-deficient cells, indicating that this defect is the result of impaired Tiam1-Rac signaling. Tiam1 interacts with Par3 and aPKCzeta, which are two components of the conserved Par3-Par6-aPKC polarity complex, and triggers biogenesis of the TJ through the activation of Rac and aPKCzeta, which is independent of Cdc42. Rac is activated upon the formation of primordial adhesions (PAs) in WT but not in Tiam1-deficient cells. Our data indicate that Tiam1-mediated activation of Rac in PAs controls TJ biogenesis and polarity in epithelial cells by association with and activation of the Par3-Par6-aPKC polarity complex.

Animals↗
PubMed2002

Casein-derived phosphopeptides disrupt tight junction integrity, and precipitously dry up milk secretion in goats.

Mammary involution is triggered by local stimuli, but the precise mechanism has not been defined. Milk stasis accumulate local signals, which makes the tight junctions (TJ) leaky. The aim of the study was to check the hypothesis that casein hydrolyzates (CNH) compromise TJ integrity and dry up milk secretion. A single dose of CNH transiently (12 to 24 h) compromised TJ integrity in the treatedudder. This was associated by a transient (12 to 96 h) decline in milk secretion. No such changes were recorded in the contralateral gland that served as a control. Four repeated doses of CNH after each milking caused drastic changes in mammary secretion and composition, which were associated with irreversible cessation of milk secretion within 96 h. No such changes were recorded in goats treated with de-phosphorylated casein (control). We conclude that CNH are the milk-borne factors that cause the disruption of TJ integrity and induction of involution, and that the serine-Ps in the CNHs are essential for the excretion of biological activity.

Animals↗
PubMed1995

EGTA-induced disruption of epithelial cell tight junctions in the lactating caprine mammary gland.

The suitability of the Ca2+ chelator ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) to induce disruption of mammary tight junctions (TJ) and its effect on milk secretion were investigated in six goats. EGTA was administered via the teat of one gland as an isosmotic (300 mosmol/l) K-EGTA solution (68 mM EGTA), whereas the control gland received an isosmotic sucrose solution. Lactose, Na, K, and Cl in milk, blood lactose, and the presence of Evans blue (EB) in mammary lymph were used as indicators of TJ disruption. EGTA caused transient (approximately 60 h) changes (P < 0.05) in the concentration of lactose, K, Na, and Cl in milk, consistent with loss of TJ integrity. This was confirmed by a rapid (< 1 h) increase (P < 0.05) in blood lactose levels. Moreover, EB appeared in lymph < 1 h after EGTA+EB treatment. Milk secretion declined unilaterally by 15% (P < 0.05) after EGTA and did not return to baseline until approximately 60 h after EGTA. EGTA caused a unilateral, temporary (first 7 h) increase in mammary blood flow. This study shows that a rapid temporary disruption of mammary TJ can be successfully induced in vivo and that such disruption compromises milk secretion.

Animals↗
PubMed2003

Hepatocyte growth factor induces MDCK cell morphogenesis without causing loss of tight junction functional integrity.

Hepatocyte growth factor (HGF) induces mitogenesis, motogenesis, and tubulogenesis of cultured Madin-Darby canine kidney (MDCK) epithelial cells. We report that in addition to these effects HGF stimulates morphogenesis of tight, polarized MDCK cell monolayers into pseudostratified layers without loss of tight junction (TJ) functional integrity. We tested TJ functional integrity during formation of pseudostratified layers. In response to HGF, the TJ marker ZO-1 remained in morphologically complete rings and functional barriers to paracellular diffusion of ruthenium red were maintained in pseudostratified layers. Transepithelial resistance (TER) increased transiently two- to threefold during the morphogenetic transition from monolayers to pseudostratified layers and then declined to baseline levels once pseudostratified layers were formed. In MDCK cells expressing the trk/met chimera, both HGF and NGF at concentrations of 2.5 ng/ml induced scattering. However, 2.5 ng/ml HGF did not affect TER. The peak effect of HGF on TER was at a concentration of 100 ng/ml. In contrast, NGF at concentrations as high as 25 microg/ml had no effect on TER or pseudostratified layer morphogenesis of trk/met-expressing cultures. These results suggest that altered presentation of the stimulus, such as through HGF interaction with low-affinity sites, may change the downstream signaling response. In addition, our results demonstrate that HGF stimulates pseudostratified layer morphogenesis while inducing an increase in TER and maintaining the overall tightness of the epithelial layer. Stimulation of epithelial cell movements by HGF without loss of functional TJs may be important for maintaining epithelial integrity during morphogenetic events such as formation of pseudostratified epithelia, organ regeneration, and tissue repair.

Animals↗
PubMed2005

Chronic inflammatory pain leads to increased blood-brain barrier permeability and tight junction protein alterations.

The blood-brain barrier (BBB) maintains brain homeostasis by limiting entry of substances to the central nervous system through interaction of transmembrane and intracellular proteins that make up endothelial cell tight junctions (TJs). Recently it was shown that the BBB can be modulated by disease pathologies including inflammatory pain. This study examined the effects of chronic inflammatory pain on the functional and molecular integrity of the BBB. Inflammatory pain was induced by injection of complete Freund's adjuvant (CFA) into the right plantar hindpaw in female Sprague-Dawley rats under halothane anesthesia; control animals were injected with saline. Edema and hyperalgesia were assessed by plethysmography and infrared paw-withdrawal latency. At 72 h postinjection, significant edema formation and hyperalgesia were noted in the CFA-treated rats. Examination of permeability of the BBB by in situ perfusion of [14C]sucrose while rats were under pentobarbital anesthesia demonstrated that CFA treatment significantly increased brain sucrose uptake. Western blot analysis of BBB TJ proteins showed no change in expression of zonula occludens-1 (an accessory protein) or actin (a cytoskeletal protein) with CFA treatment. Expression of the transmembrane TJ proteins occludin and claudin-3 and -5 significantly changed with CFA treatment with a 60% decrease in occludin, a 450% increase in claudin-3, and a 615% increase in claudin-5 expression. This study demonstrates that during chronic inflammatory pain, alterations in BBB function are associated with changes in specific transmembrane TJ proteins.

Animals↗
PubMed1998

MDCK cell cultures as an epithelial in vitro model: cytoskeleton and tight junctions as indicators for the definition of age-related stages by confocal microscopy.

PURPOSE: Madin Darby Canine Kidney (MDCK) cells were grown in culture, and age-related morphological changes in the cytoskeleton and tight junction (TJ) network were used to define stages in view of establishing an optimal in vitro model for the epithelial barrier. METHODS: Growth curves and transepithelial electrical resistance (TEER) were determined, and the cytoskeleton (actin, alpha-tubulin, vimentin) and TJ (Zonula occludens proteins ZO1, ZO2) were investigated with immunofluorescent methods by confocal laser scanning microscopy (CLSM) and digital image restoration. RESULTS: TEER measurements indicated that TJ were functional after one day. Values then remained constant. Four morphological stages could be distinguished. Stage I (0-1 day): Sub confluent cultures with flat cells; TJ established after cell-to-cell contacts are made. Stage II (2-6 days): Confluent monolayers with a complete TJ network, which remains intact throughout the later stages. Stage III (7-14 days): Rearrangement in the cytoskeleton; constant cell number; volume and surface area of cells reduced (cobble-stone appearance). Stage IV (> or = 15 days): Dome formation, i.e. thickening and spontaneous uplifting of the cell monolayer. CONCLUSIONS: Based on the structural characteristics of stage III cell cultures, which are closest to the in vivo situation, we expect them to represent an optimal in vitro model to study drug transport and/or interactions with drugs and excipients.

Animals↗
PubMed2001

Retinoid X receptor alpha and retinoic acid receptor gamma mediate expression of genes encoding tight-junction proteins and barrier function in F9 cells during visceral endodermal differentiation.

Retinoids are critical for differentiation of columnar epithelial cells and for preventing metaplasia of these cells into stratified squamous epithelial cells, in which tight junctions (TJs) are essentially absent. This implies that retinoids might play important roles in regulating the structures and functions of TJs of columnar epithelium. F9 murine embryonal carcinoma cells differentiate into epithelial cells resembling visceral endoderm bearing TJs, when grown in suspension as aggregates in the presence of retinoic acid (RA). We show that RA induces the TJ structure and expression of several TJ-associated molecules, such as ZO-1, occludin, claudin-6, and claudin-7, as well as a barrier function in the genetically engineered cell line F9:rtTA:Cre-ER(T) L32T2, which allows sophisticated genetic manipulations simply by addition of ligands (H. Chiba et al., 2000, Exp. Cell Res. 260, 334-339). Interestingly, our data indicate that a barrier for small substances is generated after that for large ones during de novo formation of TJs. We also compared the RA-induced expression of TJ components and barrier function in RXRalpha(-/-)-RARgamma(-/-) F9 cells with those in wild-type cells and show that the retinoid signals for transduction of these events are mediated by specific RXR-RAR pairs.

Animals↗
PubMedDate not supplied

Short-chain fatty acids alter tight junction permeability in intestinal monolayer cells via lipoxygenase activation.

OBJECTIVE: Involvement of lipoxygenase (LOX) and cyclo-oxygenase (COX) on cellular differentiation or apoptosis induced by butyrate has been reported recently, but the effect on tight junction (TJ) permeability has not been reported. One major activity of butyrate and, to a lesser extent, propionate is to modulate gene transcription via histone acetylation by their histone deacetylase inhibitor activity. In this study, we evaluated the activation of LOX and COX in TJ permeability changes by short-chain fatty acids, butyrate, propionate, and acetate in intestinal monolayer cells and their possible mechanism by histone acetylation. METHODS: The effects of LOX and COX inhibitors on TJ permeability and the expression of LOX or COX mRNA induced by short-chain fatty acids were investigated in Caco-2 cells using Transwell chambers. The effects of hydroxyeicosatetraenoic acid (a product of LOX) on TJ permeability were also evaluated. The effects of short-chain fatty acids were compared with those of trichostatin A (histone deacetylase inhibitor). RESULTS: A LOX inhibitor clearly inhibited the effect of butyrate on TJ permeability, whereas COX inhibitors did not. The LOX and COX inhibitors partly inhibited the effects of propionate but not of acetate. Butyrate increased LOX mRNA expression, and hydroxyeicosatetraenoic acid and trichostatin A mimicked its effect. CONCLUSION: These results suggest that short-chain fatty acids, especially butyrate, induce TJ permeability changes through LOX activation through histone acetylation.

Acetylation↗
PubMed2004

Increased iNOS activity is essential for intestinal epithelial tight junction dysfunction in endotoxemic mice.

We tested the hypothesis that increased production of nitric oxide (NO.) associated with lipopolysaccharide (LPS)-induced systemic inflammation leads to functionally significant alterations in the expression and/or targeting of key tight junction (TJ) proteins in ileal and colonic epithelium. Wild-type or inducible NO. synthase (iNOS) knockout male C57B1/6J mice were injected intraperitoneally with 2 mg/kg Escherichia coli O111:B4 LPS. iNOS was inhibited using intraperitoneal L-N(6)-(1-iminoethyl)lysine (L-NIL; 5 mg/kg). Immunoblotting of total protein and NP-40 insoluble proteins revealed decreased expression and decreased TJ localization, respectively, of the TJ proteins, zonula occludens (ZO)-1, ZO-2, ZO-3, and/or occludin in ileal mucosa and colonic mucosa (total protein only) after injection of C57B1/6J mice with LPS. Immunohistochemistry showed deranged distribution of ZO-1 and occludin in both tissues from endotoxemic mice. Endotoxemia was associated with evidence of gut epithelial barrier dysfunction evidenced by increased ileal mucosal permeability to fluorescein isothiocyanate-dextran (Mr=4 kDa) and increased bacterial translocation to mesenteric lymph nodes. Pharmacologic inhibition of iNOS activity using L-NIL or genetic ablation of the iNOS gene ameliorated LPS-induced changes in TJ protein expression and gut mucosal barrier function. These results support the view that at least one mechanism contributing to the pathogenesis of gastrointestinal epithelial dysfunction secondary to systemic inflammation is increased iNOS-dependent NO. production leading to altered expression and localization of key TJ proteins.

Animals↗