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Apical and basolateral cell membranes are separated by tight junctions (TJs). Microvilli are limited to the apical cell membrane. TJs and microvilli are the landmarks for epithelial cell polarity. However, the direct relationship between TJ proteins (TJPs) and the components of microvilli remains unclear. In this study, we investigated whether occludin, which is considered to be a functional TJP, is involved in microvillus formation. In occludin knockout mouse hepatic cells (OcKO cells), the microvillus density was less than that in wild-type (WT) cells and the length of microvilli was short. Immunoreactivity of ezrin was decreased in OcKO cells compared with that in WT cells. Although there was no change in the expression level of ezrin, phosphorylation of ezrin was decreased in OcKO cells. The microvillus density and the length of microvilli were increased in OcKO cells by transfection of full-length mouse occludin and COOH-terminal domains of occludin. These results suggested that occludin induced microvillus formation via phosphorylation of ezrin and that the COOH-terminal domain of occludin, which is localized in non-TJ areas, might be able to induce microvilli formation. Our results provide new insights into the function of occludin., (Copyright © 2018 Elsevier Inc. All rights reserved.)

c-Jun N-terminal kinase (JNK), known as a stress-activated protein kinase, regulates normal epithelial biological processes, including assembly of adherens and tight junctions, and it is involved in the development of several cancers. The JNK inhibitor SP600125 enhances epithelial barrier function through modulation of tight junction molecules in normal human pancreatic epithelial cells. Furthermore, this JNK inhibitor suppresses the growth of human pancreatic cancer cells. However, the effects of SP600125 on the epithelial barrier in human pancreatic cancer cells remain unknown. In the present study, the JNK inhibitor SP600125 markedly enhanced the barrier function and cell elongation of well-differentiated human pancreatic cancer cell line HPAC in a Ca-switch model. The epithelial barrier function induced by SP600125 was regulated by phosphorylated β-catenin without changes in the tight junction molecules. The cell elongation induced by SP600125 was closely related to the expression of the F-actin-binding protein DrebrinE. These findings suggest that JNK is involved in the regulation of the epithelial barrier function and cell shape during remodeling of pancreatic cancer cells. The JNK inhibitor SP600125 may have potential as a therapeutic drug for pancreatic cancer via induction of differentiation.

Pancreatic cancer continues to be a leading cause of cancer-related death worldwide and there is an urgent need to develop novel diagnostic and therapeutic strategies to reduce the mortality of patients with this disease. In pancreatic cancer, some tight junction proteins, including claudins, are abnormally regulated and therefore are promising molecular targets for diagnosis, prognosis and therapy. Claudin-4 and -18 are overexpressed in human pancreatic cancer and its precursor lesions. Claudin-4 is a high affinity receptor of Clostridium perfringens enterotoxin (CPE). The cytotoxic effects of CPE and monoclonal antibodies against claudin-4 are useful as novel therapeutic tools for pancreatic cancer. Claudin-18 could be a putative marker and therapeutic target with prognostic implications for patients with pancreatic cancer. Claudin-1, -7, tricellulin and marvelD3 are involved in epithelial to mesenchymal transition (EMT) of pancreatic cancer cells and thus might be useful as biomarkers during disease. Protein kinase C is closely related to EMT of pancreatic cancer and regulates tight junctions of normal human pancreatic duct epithelial cells and the cancer cells. This review focuses on the regulation of tight junctions via protein kinase C during EMT in human pancreatic cancer for the purpose of developing new diagnostic and therapeutic modalities for pancreatic cancer.

Background: Pseudomonas aeruginosa causes chronic respiratory disease, and the elastase enzyme that it produces increases the permeability of airway epithelial cells owing to the disruption of tight junctions. P. aeruginosa is also implicated in prolonged chronic rhinosinusitis. However, the effects of P. aeruginosa elastase (PE) against the barrier formed by human nasal epithelial cells (HNECs) remain unknown., Methods: To investigate the mechanisms involved in the disruption of tight junctions by PE in HNECs, primary cultures of HNECs transfected with human telomerase reverse transcriptase (hTERT-HNECs) were used. The hTERT-HNECs were pretreated with inhibitors of various signal transduction pathways, PKC, MAPK, p38MAPK, PI3K, JNK, NF-κB, EGF receptor, proteasome, COX1 and COX2 before treatment with PE. Some cells were pretreated with siRNA and agonist of protease activated receptor-2 (PAR-2) before treatment with PE. Expression and structures of tight junctions were determined by Western blotting, real-time PCR, immunostaining and freeze-fracture. Transepithelial electrical resistance (TER) was examined as the epithelial barrier function., Results: PE treatment transiently disrupted the epithelial barrier and downregulated the transmembrane proteins claudin-1 and -4, occludin, and tricellulin, but not the scaffold PDZ-expression proteins ZO-1 and -2 and adherens junction proteins E-cadherin and β-catenin. The transient downregulation of tight junction proteins was controlled via distinct signal transduction pathways such as the PKC, MAPK, PI3K, p38 MAPK, JNK, COX-1 and -2, and NF-κB pathways. Furthermore, treatment with PE transiently decreased PAR-2 expression, which also regulated the expression of the tight junction proteins. Treatment with a PAR-2 agonist prevented the downregulation of the tight junction proteins after PE treatment in HNECs., Conclusions: PE transiently disrupts tight junctions in HNECs and downregulates PAR-2. The transient disruption of tight junctions by PE might occur repeatedly during chronic rhinosinusitis.

The number of patients with uterine endometrial carcinoma, the cause of which involves sex hormones, has recently been growing rapidly because of increases in life expectancy and obesity. Tight junction proteins claudin-3 and -4 are receptors of Clostridium perfringens enterotoxin (CPE) and increase during endometrial carcinogenesis. In the present study of normal human endometrial epithelial (HEE) cells and the uterus cancer cell line Sawano, we investigate changes in the expression of tight junction proteins including claudin-3 and -4, the fence and barrier functions of the tight junction and the cytotoxic effects of CPE by sex hormones. In primary cultured HEE cells, treatment with progesterone (P4) but not estradiol (E2), induced claudin-1, -3, -4 and -7 and occludin, together with the downregulation of the barrier function but not the fence function. In Sawano cells, claudin-3 and -4 were upregulated by E2 but not by P4, together with a disruption of both the barrier and fence function. In primary cultured HEE cells, claudin-3 and -4 were localized at the apicalmost regions (tight junction areas) and no cytotoxicity of CPE was observed. In Sawano cells, claudin-3 and -4 were found not only in the apicalmost regions but also at the basolateral membrane and the cytotoxicity of CPE was enhanced by E2. Thus, tight junctions are physiological regulated by sex hormones in normal HEE cells during the menstrual cycle suggesting that safer and more effective therapeutic methods targeting claudins in uterine cancer can be developed.

Tight junctions are intercellular junctions adjacent to the apical ends of paracellular spaces. They have two classical functions, the barrier function and the fence function. The former regulates the passage of ions, water and various molecules through paracellular spaces, and is thus related to edema, jaundice, diarrhea and blood-borne metastasis. The latter function maintains cell polarity by forming a fence to prevent intermixing of molecules in the apical membrane with those in the lateral membrane. This function is deeply involved in cancer cell properties in terms of loss of cell polarity. Recently, two novel aspects of tight junctions have been reported. One is their involvement in signal transduction. The other is that fact that tight junctions are considered to be a crucial component of innate immunity. In addition, since some proteins comprising tight junctions work as receptors for viruses and extracellular stimuli, pathogenic bacteria and viruses target and affect the tight junction functions, leading to diseases. In this review, the relationship between tight junctions and human diseases will be described., (© 2012 The Author. Pathology International © 2012 Japanese Society of Pathology and Wiley Publishing Asia Pty Ltd.)

The mucosal barrier of the upper respiratory tract including the nasal cavity, which is the first site of exposure to inhaled antigens, plays an important role in host defense in terms of innate immunity and is regulated in large part by tight junctions of epithelial cells. Tight junction molecules are expressed in both M cells and dendritic cells as well as epithelial cells of upper airway. Various antigens are sampled, transported, and released to lymphocytes through the cells in nasal mucosa while they maintain the integrity of the barrier. Expression of tight junction molecules and the barrier function in normal human nasal epithelial cells (HNECs) are affected by various stimuli including growth factor, TLR ligand, and cytokine. In addition, epithelial-derived thymic stromal lymphopoietin (TSLP), which is a master switch for allergic inflammatory diseases including allergic rhinitis, enhances the barrier function together with an increase of tight junction molecules in HNECs. Furthermore, respiratory syncytial virus infection in HNECs in vitro induces expression of tight junction molecules and the barrier function together with proinflammatory cytokine release. This paper summarizes the recent progress in our understanding of the regulation of tight junctions in the upper airway epithelium under normal, allergic, and RSV-infected conditions.

Tricellulin is an important component of tricellular tight junctions (TJs) and is involved in the formation of tricellular contacts. However, little is known about its regulation during the assembly and disassembly of tricellular TJs. By using the well-differentiated pancreatic cancer cell line HPAC, which highly expresses tricellulin at tricellular contacts, we have investigated changes in the localization, expression and phosphorylation of tricellulin and in its TJ functions as a barrier and fence during the destruction and formation of TJs induced by changes in the extracellular calcium concentration. During both extracellular Ca(2+) depletion caused by EGTA treatment and Ca(2+) repletion after Ca(2+) starvation, the expression of tricellulin increased in whole lysates and in Triton-X-100-insoluble fractions without any change in its mRNA. The increases in immunoreactivity revealed by Western blotting were prevented by alkaline phosphatase treatment. Immunoprecipitation assays showed that tricellulin was phosphorylated on threonine residues when it increased after Ca(2+) depletion and repletion. In the early stage after Ca(2+) repletion, tricellulin was expressed not only at tricellular contacts but also in the cytoplasm and at bicellular borders. In confocal laser microscopy, tricellulin was observed at the apical-most regions and basolateral membranes of tricellular contacts after Ca(2+) repletion. Knockdown of tricellulin delayed the recovery of the barrier and fence functions after Ca(2+) repletion. Thus, the dynamic behavior of tricellulin during the destruction and formation of TJs under various extracellular calcium conditions seems to be closely associated with the barrier and fence functions of TJs.

Ovarian adenocarcinomas, like human ovarian surface epithelial cells, form functional tight junctions. Tight junction molecules claudin-3 and claudin-4, which are the receptors of Clostridium perfringens enterotoxin (CPE), are abnormally upregulated in epithelial ovarian cancers of all subtypes including, mucinous cystadenocarcinoma and serous cystadenocarcinoma. Clostridium perfringens enterotoxin may be a novel tumor-targeted therapy for ovarian cancers. In epithelial ovarian cancers, overexpression of epidermal growth factor receptor has been observed and the exogenous ligand EGF induces epithelial-mesenchymal transition in ovarian surface epithelium. Epidermal growth factor (EGF) signaling modulates expression of claudins with changes of fence and barrier functions in various cell types. However, the regulation of tight junctions by EGF in ovarian cancers remains unclear. In the present study, to investigate the mechanisms of the regulation of tight junctions in ovarian cancers, ovarian cancer cell lines mucinous cystadenocarcinoma (MCAS) and serous cystadenocarcinoma (HUOA) were treated with EGF. Epidermal growth factor downregulated claudin-3 in MCAS and claudin-4 in HUOA by inducing degradation of the proteins with changes in structures and functions of tight junctions via the MEK/ERK or PI3K/Akt signaling pathway. In addition, in HUOA but not MCAS, EGF downregulated the cytotoxic effect of CPE via claudin-4. Thus, there were different mechanisms for regulation of claudins by EGF between subtypes of epithelial ovarian cancer cells in vitro. These results indicate that EGF may affect claudins and tight junctional functions in ovarian cancer cells during cancer progression.

To investigate the regulation of tight junction molecules in normal human pancreatic duct epithelial (HPDE) cells and pancreatic cancer cells, we introduced the human telomerase reverse transcriptase (hTERT) gene into HPDE cells in primary culture and compared them to pancreatic cancer cell lines. The hTERT-transfected HPDE cells were positive for PDE markers and expressed claudin-1, claudin-4, claudin-7, and claudin-18, occludin, tricellulin, marvelD3, JAM-A, zonula occludens (ZO)-1, and ZO-2. The tight junction molecules, including claudin-4 and claudin-18 of normal HPDE cells, were in part regulated via a protein kinase C signal pathway by transcriptional control. In addition, claudin-18 in normal HPDE cells and pancreatic cancer cells was markedly induced by a PKC activator, and claudin-18 in pancreatic cancer cells was also modified by DNA methylation. In the marvel family of normal HPDE cells and pancreatic cancer cells, tricellulin was upregulated via a c-Jun N-terminal kinase pathway, and marvelD3 was downregulated during Snail-induced epithelial-mesenchymal transition., (© 2012 New York Academy of Sciences.)

The novel tight junction protein marvelD3 contains a conserved MARVEL (MAL and related proteins for vesicle trafficking and membrane link) domain like occludin and tricellulin. However, little is yet known about the detailed role and regulation of marvelD3 in normal epithelial cells and cancer cells, including pancreatic cancer. In the present study, we investigated marvelD3 expression in well and poorly differentiated human pancreatic cancer cell lines and normal pancreatic duct epithelial cells in which the hTERT gene was introduced into human pancreatic duct epithelial cells in primary culture, and the changes of marvelD3 during Snail-induced epithelial-mesenchymal transition (EMT) under hypoxia, TGF-β treatment and knockdown of FOXA2 in well differentiated pancreatic cancer HPAC cells. MarvelD3 was transcriptionally downregulated in poorly differentiated pancreatic cancer cells and during Snail-induced EMT of pancreatic cancer cells in which Snail was highly expressed and the fence function downregulated, whereas it was maintained in well differentiated human pancreatic cancer cells and normal pancreatic duct epithelial cells. Depletion of marvelD3 by siRNAs in HPAC cells resulted in downregulation of barrier functions indicated as a decrease in transepithelial electric resistance and an increase of permeability to fluorescent dextran tracers, whereas it did not affect fence function of tight junctions. In conclusion, marvelD3 is transcriptionally downregulated in Snail-induced EMT during the progression for the pancreatic cancer., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Tight junctions of hepatocytes play crucial roles in the barrier to keep bile in bile canaliculi away from the blood circulation, which we call the blood-biliary barrier. Tight junction proteins of hepatocytes are regulated by various cytokines and growth factors via distinct signal transduction pathways. To investigate changes in expression and function of tight junction proteins including claudins via signal transduction pathways in hepatocytes during EMT induced by TGF-β, we examined effects of TGF-β on expression and localization of the integral tight junction proteins, claudin-1, -2, and occludin, as well as the fence function by using primary cultures of adult rat hepatocytes.

Respiratory syncytial virus (RSV) primarily infects upper respiratory tract cells, mainly nasal epithelial cells. The tight junctions of nasal epithelial cells are thought to perform important innate immune function against foreign materials including respiratory viruses. We investigated in vitro the relationship of RSV infection and the tight junctions of primary nasal epithelial cells which had been transfected with human telomerase reverse transcriptase (hTERT) to prolong cell life. Nasal epithelial cells developed tight junctions when cultured in medium containing fetal bovine serum, and these cells showed apparent resistance to RSV infection compared to control cells. RSV could infect these cells from apical but not basolateral side, suggesting that only apical side possess RSV receptor or a mechanism for absorbing RSV particles. Importantly, RSV infection of the cells enhanced the expression of tight junction proteins occludin, claudin-4 and ZO-1. These findings suggest that RSV infection induces polarity in the infected cells. This polarity could facilitate cellular secretion of propagated RSV, thereby spreading the infection., (Copyright © 2011 S. Karger AG, Basel.)

Autotypic tight junctions are formed by tight junction-like structures in three regions of myelinating Schwann cells, the paranodal loops, Schmidt-Lanterman incisures, and outer/inner mesaxons, and various tight junction molecules, including claudin-19 and junctional adhesion molecule (JAM)-C. Our findings demonstrate the identification and subcellular distribution of a novel tricellular tight junction protein, tricellulin (TRIC), in the autotypic tight junctions of mouse myelinating Schwann cells, compared with the autotypic adherens junction protein E-cadherin and the autotypic tight junction protein JAM-C, which are expressed in the paranodal loops, Schmidt-Lanterman incisures, and mesaxons. In real-time RT-PCR, the expression level of TRIC mRNA was about 10-fold higher in the sciatic nerve than in the spinal cord or cerebrum. In immunostaining, TRIC signals were completely restricted to the peripheral nervous system (PNS) and strongly concentrated at the paranodal loops, Schmidt-Lanterman incisures, and mesaxons of myelinating Schwann cells. In addition, TRIC was expressed in the thin region of the paranode and there was a gap between TRIC and the Na+ channel. Furthermore, TRIC was more distally located from the node than E-cadherin and was colocalized with JAM-C. It is possible that TRIC may be a component to maintain the integrity for PNS myelin function and morphology. This manuscript contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials.

Tricellulin (TRIC) is a tight junction protein at tricellular contacts where three epithelial cells meet, and it is required for the maintenance of the epithelial barrier. To investigate whether TRIC is regulated via a c-Jun N-terminal kinase (JNK) pathway, human pancreatic HPAC cells, highly expressed at tricellular contacts, were exposed to various stimuli such as the JNK activators anisomycin and 12-O-tetradecanoylphorbol 13-acetate (TPA), and the proinflammatory cytokines IL-1β, TNFα, and IL-1α. TRIC expression and the barrier function were moderated by treatment with the JNK activator anisomycin, and suppressed not only by inhibitors of JNK and PKC but also by siRNAs of TRIC. TRIC expression was induced by treatment with the PKC activator TPA and proinflammatory cytokines IL-1β, TNFα, and IL-1α, whereas the changes were inhibited by a JNK inhibitor. Furthermore, in normal human pancreatic duct epithelial cells using hTERT-transfected primary cultured cells, the responses of TRIC expression to the various stimuli were similar to those in HPAC cells. TRIC expression in tricellular tight junctions is strongly regulated together with the barrier function via the JNK transduction pathway. These findings suggest that JNK may be involved in the regulation of tricellular tight junctions including TRIC expression and the barrier function during normal remodeling of epithelial cells, and prevent disruption of the epithelial barrier in inflammation and other disorders in pancreatic duct epithelial cells., (© 2010 Wiley-Liss, Inc.)

In human pancreatic cancer, integral membrane proteins of tight junction claudins are abnormally regulated, making these proteins promising molecular diagnostic and therapeutic targets. However, the regulation of claudin-based tight junctions remains unknown not only in the pancreatic cancer cells but also in normal human pancreatic duct epithelial (HPDE) cells. To investigate the regulation of tight junction molecules including claudins in normal HPDE cells, we introduced the human telomerase reverse transcriptase (hTERT) gene into HPDE cells in primary culture. The hTERT-transfected HPDE (hTERT-HPDE) cells were positive for the pancreatic duct epithelial markers such as CK7, CK19, and carbonic anhydrase isozyme 2 and expressed epithelial tight junction molecules claudin-1, -4, -7 and, -18, occludin, JAM-A, ZO-1, ZO-2, and tricellulin. By treatment with fetal bovine serum or 12-O-tetradecanoylphorbol 13-acetate (TPA), the tight junction molecules were up-regulated at the transcriptional level via a protein kinase C (PKC) signal pathway. A PKC-alpha inhibitor, Gö6976, prevented up-regulation of claudin-4 by TPA. Furthermore, a PKC-delta inhibitor, rottlerin, prevented up-regulation of claudin-7, occludin, ZO-1, and ZO-2 by TPA. By GeneChip analysis, up-regulation of the transcription factor ELF3 was observed in both fetal bovine serum- and TPA-treated cells. Treatment with small interfering RNAs of ELF3 prevented up-regulation of claudin-7 by TPA. These data suggest that tight junctions of normal HPDE cells were at least in part regulated via a PKC signal pathway by transcriptional control.

Peroxisome proliferator activated (PPAR)gamma plays a critical role in the control of not only adipocyte differentiation, lipid metabolism and immunity but also the barrier functions of epithelial and endothelial cells. In the present study, to investigate effects of PPAR gamma agonists on the tight junctional barrier of human nasal epithelial cells (HNECs), hTERT-transfected HNECs, which highly express both PPAR gamma and tight junction proteins, were treated with the PPAR gamma agonists rosiglitazone and troglitazone. Treatment with the PPAR gamma agonists enhanced the barrier function of hTERT-transfected HNECs together with the upregulation of tight junction molecules claudin-1 and -4, occludin, and tricellulin at the transcriptional level. A significant increase of tight junction strands was also observed after treatment with rosiglitazone. Treatment with PPAR gamma agonists induced the activity of phospho-PKC in hTERT-transfected HNECs. The upregulation of the tight junction molecules in hTERT-transfected HNECs by rosiglitazone was inhibited by not only PPAR gamma antagonists GW9662 and T0070907, but also the panPKC inhibitor GF109203X. These findings suggest that PPAR gamma agonists upregulate the barrier function of tight junctions of human nasal epithelial cells via a PKC signaling pathway and could be novel drugs for protection against inhaled substances and pathogens in the airway epithelium of human nasal mucosa., ((c) 2010 Elsevier Ltd. All rights reserved.)

Epithelial-derived thymic stromal lymphopoietin (TSLP) is an IL-7-like cytokine that triggers dendritic cell (DC)-mediated Th2-type inflammatory responses. The activated DCs can penetrate the epithelium to directly take up antigen without compromising the barrier function. Although it is reported that DCs express tight junction molecules and can establish tight junction-like structures with adjacent epithelial cells to preserve the epithelial barrier, the regulation of expression of tight junction molecules in DCs remains unknown. In the present study, to investigate the mechanical regulation of expression of tight junction molecules in DCs, XS52 DCs that was a long-term DC line established from the epidermis of a newborn BALB/c mouse, were treated with TSLP or toll-like receptor (TLR) ligands. In XS52 cells, tight junction molecules claudin-1, -3, -4, -6, -7, -8, and occludin were detected. mRNA expression of TSLP receptor and all these tight junction molecules was significantly increased in activated XS52 cells after treatment with TSLP. In addition, expression of claudin-7 protein was increased in dose- and time-dependent manner. In XS52 cells, which express TLR2, TLR3, TLR4, and TLR7, but not TLR9, expression of claudin-7 protein was also increased after treatment with ligands of TLR2, TLR4 or TLR7/8, Pam3Cys-Ser-(Lys)4, LPS, or CL097. The NF-kappaB inhibitor IMD-0354 prevented upregulation of claudin-7 after treatment with TSLP or TLR ligands. These findings indicate that TSLP induces expression of tight junction protein claudin-7 in DCs via NF-kappaB as well as via TLRs and may control tight junctions of DCs to preserve the epithelial barrier during allergic inflammation.

Tricellulin was identified as the first marker of the tricellular tight junction, which forms at the meeting points of three cells, and it is required for the maintenance of the transepithelial barrier. Although it is also considered to be important for the mucosal barrier of the upper respiratory tract, little is known about its expression and localization. In the present study, we examined the expression and localization of tricellulin in normal human nasal epithelial cells in vivo and in vitro, especially using primary cultures and telomerase reverse transcriptase (hTERT)-transfected cells. In human nasal epithelial cells in vivo and in vitro, mRNA and protein of tricellulin were detected. It was localized not only at tricellular contacts but also at bicellular borders, and in part colocalized with occludin. In human nasal epithelium, by immunoelectron microscopy analysis, tricellulin-associated gold particles were observed around the junction-like structure of the uppermost region. By treatment with 10% fetal bovine serum (FBS), expression of tricellulin mRNA was weakly increased, whereas that of bicellular tight junction molecules was strongly increased, in real-time PCR. These results suggest that tricellulin is stably expressed in human nasal epithelial cells and may play an important role for the sealing of the corner at tricellular contacts to prevent infiltration by various inhaled viruses and antigens.

Epithelial-derived thymic stromal lymphopoietin (TSLP) triggers dendritic cell (DC)-mediated Th2-type inflammatory responses and is a master switch for allergic inflammatory diseases. In the present study, the expression and induction of TSLP and the effects of TSLP on the tight-junctional barrier of human nasal epithelial cells (HNECs) have been investigated in order to elucidate the role of TSLP in allergic rhinitis. We have found high expression of TSLP in the epithelium from patients with allergic rhinitis with recruitment and infiltration of DCs. In vitro, TSLP is significantly produced in HNECs after treatment with a toll-like receptor 2 (TLR2) ligand, Pam(3)Cys-Ser-(Lys)(4), and a mixture of interleukin-1beta and tumor necrosis factor-alpha. Treatment with TSLP rapidly enhances the barrier function of cultured HNECs, together with an increase of tight-junction proteins claudin-1, -4, -7, and occludin. The nasal-epithelial-derived TSLP thus not only activates DCs but also preserves the epithelial barrier via the upregulation of tight-junction proteins, thereby regulating antigen sensitization during the early stage of allergic rhinitis.

Tight junctions of hepatocytes play crucial roles in the barrier to keep bile in bile canaliculi away from the blood circulation, which we call the blood-billiary-barrier (Kojima et al., 2003). Tight junction proteins of hepatocytes are regulated by various cytokines and growth factors via distinct signal transduction pathways. They are also considered to participate in signal transduction pathways that regulate epithelial cell proliferation, gene expression, differentiation and morphogenesis. This review focuses on recent findings about the relationship between tight junction proteins and signal transduction pathways in hepatocytes.

The epithelium of upper respiratory tissues such as human nasal mucosa forms a continuous barrier via tight junctions, which is thought to be regulated in part through a protein kinase C (PKC) signaling pathway. To investigate the mechanisms of the regulation of PKC-mediated tight junction barrier function of human nasal epithelium in detail, primary human nasal epithelial cells were treated with the PKC activator 12-O-tetradecanoylophorbol-13-acetate (TPA). In primary human nasal epithelial cells, treatment with TPA led not only to activation of phosphorylation of PKC, myristoylated alanine-rich C kinase substrate, and mitogen-activated protein kinase but also expression of novel PKC-delta, PKC-theta, and PKC-epsilon. Treatment with TPA increased transepithelial electrical resistance, with tight junction barrier function more than 4-fold that of the control, together with up-regulation of tight junction proteins, occludin, zona occludens (ZO)-1, ZO-2 and claudin-1 at the transcriptional level. Furthermore, it affected the subcellular localization of the tight junction proteins and the numbers of tight junction strands. The up-regulation of barrier function and tight junction proteins was prevented by a pan-PKC inhibitor, and the inhibitors of PKC-delta and PKC-theta but not PKC-epsilon. In primary human nasal epithelial cells, transcriptional factors GATA-3 and -6 were detected by reverse transcription-polymerase chain reaction. The knockdown of GATA-3 using RNA interference resulted in inhibition of up-regulation of ZO-1 and ZO-2 by treatment with TPA. These results suggest that TPA-induced PKC signaling enhances the barrier function of human nasal epithelial cells via transcriptional up-regulation of tight junction proteins, and the mechanisms may contribute to a drug delivery system.

The human adenoid epithelium forms a continuous barrier against a wide variety of exogenous antigens. In this study, to elucidate the structures of the epithelial barrier in the human adenoid, including M-cells, we identified M-cells using an anti-cytokeratin 20 (Ck20) antibody and investigated expression of tight junction proteins in human adenoid epithelium in vivo and in vitro. In human adenoid epithelium and primary cultures, mRNAs of occludin, junctional adhesion molecule-A, ZO-1, and claudin-1, -4, -7, and -8 were detected by reverse transcription-polymerase chain reaction, whereas claudin-2 and -9 were expressed in vitro. In the epithelium in vivo, some Ck20-positive cells were randomly observed and indicated pocket-like structures, whereas Ck7 was positive in almost cells. Transmission electron microscopy revealed that Ck20-associated gold particles could be identified in M-like cells which had short microvilli and harboured the lymphocyte in the pocket-like structure. In primary cultures in vitro, Ck20-positive cells were also detected and had a function to take up fluorescent microparticles. In Ck20-positive cells in vivo and in vitro, expression of occludin, ZO-1, claudin-1 and -7 were observed at cell borders. These results indicate that the epithelial barrier of the human adenoid is stably maintained by expression of tight junction proteins in the epithelium including Ck20-positive M-like cells.

Ca(2+) is absorbed across intestinal epithelial monolayers via transcellular and paracellular pathways, and an active form of vitamin D(3), 1alpha,25-dihydroxyvitamin D(3) [1alpha,25(OH)(2)D(3)], is known to promote intestinal Ca(2+) absorption. However, the molecules driving the paracellular Ca(2+) absorption and its vitamin D dependency remain obscure. Because the tight junction proteins claudins are suggested to form paracellular channels for selective ions between neighboring cells, we hypothesized that specific intestinal claudins might facilitate paracellular Ca(2+) transport and that expression of these claudins could be induced by 1alpha,25(OH)(2)D(3). Herein, we show, by using RNA interference and overexpression strategies, that claudin-2 and claudin-12 contribute to Ca(2+) absorption in intestinal epithelial cells. We also provide evidence showing that expression of claudins-2 and -12 is up-regulated in enterocytes in vitro and in vivo by 1alpha,25(OH)(2)D(3) through the vitamin D receptor. These findings strongly suggest that claudin-2- and/or claudin-12-based tight junctions form paracellular Ca(2+) channels in intestinal epithelia, and they highlight a novel mechanism behind vitamin D-dependent calcium homeostasis.

Background/aims: Transforming growth factor-beta (TGF-beta) initiates and maintains epithelial-mesenchymal transition (EMT), which causes disassembly of tight junctions and loss of epithelial cell polarity. In mature hepatocytes during EMT induced by TGF-beta, changes in the expression of tight junction proteins and the fence function indicated that epithelial cell polarity remains unclear., Methods: In the present study, using primary cultures of adult rat hepatocytes at day 10 after plating, in which epithelial cell polarity is well maintained by tight junctions, we examined the effects of 0.01-20 ng/ml TGF-beta on the expression of the integral tight junction proteins, claudin-1, -2 and occludin, as well as the fence function., Results: In adult rat hepatocytes, TGF-beta induced EMT, which was indicated as upregulation of Smad-interacting protein-1 (SIP1) and Snail and down-regulation of E-cadherin. Down-regulation of claudin-1 and upregulation of occludin were observed beginning from a low dose of TGF-beta, whereas upregulation of claudin-2 was observed at a high dose of TGF-beta. Furthermore, treatment with TGF-beta caused disruption of the fence function, which was closely associated with the expression of claudin-1 via p38 mitogen-activated protein kinase (MAPK), phosphoinositide-3 kinase and protein kinase C but not MAPK signalling pathways., Conclusion: These results suggest that in mature hepatocytes in vitro, TGF-beta induces EMT by down-regulation of claudin-1 and the fence function via distinct signalling pathways.

Tight junctions contribute to the paracellular barrier, the fence dividing plasma membranes, and signal transduction, acting as a multifunctional complex in vertebrate epithelial and endothelial cells. The identification and characterization of the transmembrane proteins of tight junctions, claudins, junctional adhesion molecules (JAMs), occludin and tricellulin, have led to insights into the molecular nature of tight junctions. We provide an overview of recent progress in studies on these proteins and highlight their roles and regulation, as well as their functional significance in human diseases.

The epithelium of upper respiratory tissues such as nasal mucosa forms a continuous barrier to a wide variety of exogenous antigens. The epithelial barrier function is regulated in large part by the intercellular junctions, referred to as gap and tight junctions. However, changes of gap and tight junctions during differentiation of human nasal epithelial (HNE) cells are still unclear. In the present study, to investigate changes of gap and tight junctions during differentiation of HNE cells in vitro, we used primary human HNE cells cocultured with primary human nasal fibroblast (HNF) cells in a noncontact system. In HNE cells cocultured with HNF cells for 2 weeks, numerous elongated cilia-like structures were observed compared to those without HNF cells. In the coculture, downregulation of Cx26 and upregulation of Cx30.3 and Cx31 were observed together with extensive gap junctional intercellular communication. Furthermore, expression of the tight junction proteins claudin-1, claudin-4, occludin and ZO-2 was increased. These results suggest that switching in expression of connexins and induction of tight junction proteins may be closely associated with differentiation of HNE cells in vitro and that differentiation of HNE cells requires unknown soluble factors secreted from HNF cells.

Connexins (Cx) are considered to play a crucial role in the differentiation of epithelial cells and to be associated with adherens and tight junctions. This review describes how connexins contribute to the induction and maintenance of tight junctions in epithelial cells, hepatic cells and airway epithelial cells. Endogenous Cx32 expression and mediated intercellular communication are associated with the expression of tight junction proteins of primary cultured rat hepatocytes. We introduced the human Cx32 gene into immortalized mouse hepatic cells derived from Cx32-deficient mice. Exogenous Cx32 expression and the mediated intercellular communication by transfection could induce the expression and function of tight junctions. Transfection also induced expression of MAGI-1, which localized at adherens and tight junction areas in a gap junctional intercellular communication (GJIC)-independent manner. Furthermore, expression of Cx32 was related to the formation of single epithelial cell polarity of the hepatic cells. On the other hand, Cx26 expression, but not mediated intercellular communication, contributed to the expression and function of tight junctions in human airway epithelial cells. We introduced the human Cx26 gene into the human airway epithelial cell line Calu-3 and used a model of tight junction disruption by the Na(+)/K(+)-ATPase inhibitor ouabain. Transfection with Cx26 prevented disruption of both tight junction functions, the fence and barrier, and the changes of tight junction proteins by treatment with ouabain in a GJIC-independent manner. These results suggest that connexins can induce and maintain tight junctions in both GJIC-dependent and -independent manners in epithelial cells.

In rodent livers, integral tight junction (TJ) proteins claudin-1, -2, -3, -5 and -14 are detected and play crucial roles in the barrier to keep bile in bile canaculi away from the blood circulation. Claudin-2 shows a lobular gradient increasing from periportal to pericentral hepatocytes, whereas claudin-1 and -3 are expressed in the whole liver lobule. Although claudin-2 expression induces cation-selective channels in tight junctions of epithelial cells, the physiological functions and regulation of claudin-2 in hepatocytes remain unclear. Oncostatin M (OSM) is a multifunctional cytokine implicated in the differentiation of hepatocytes that induces formation of E-cadherin-based adherens junctions in fetal hepatocytes. In this study, we examined whether OSM could induce expression and function of claudin-2 in rodent hepatocytes, immortalized mouse and primary cultured proliferative rat hepatocytes. In the immortalized mouse and primary cultured proliferative rat hepatocytes, treatment with OSM markedly increased mRNA and protein of claudin-2 together with formation of developed networks of TJ strands. The increase of claudin-2 enhanced the paracellular barrier function which depended on molecular size. The increase of claudin-2 expression induced by OSM in rodent hepatocytes was regulated through distinct signaling pathways including PKC. These results suggest that expression of claudin-2 in rodent hepatocytes may play a specific role as controlling the size of paracellular permeability in the barrier to keep bile in bile canaculi.

Claudins are the transmembrane proteins forming the backbone of tight junctions, and consist of over 20 members of a gene family. Claudins are expressed in a tissue- and cell-type specific fashion, and changes in their abundance and/or distribution are proposed to play important roles in the pathophysiology of numerous disorders. In the prostate, claudin-1, -3, -4 and -7 transcripts are known to be expressed, but it is unknown regarding mRNA expression of other claudins or concerning expression and localization of claudin proteins in this organ. We herein show, by RT-PCR and Western blotting analyses, that not only these four claudins but also claudin-5, -8 and -10 are expressed in the normal mouse prostate. Claudin-3, -4, -5, -8 and -10 were primarily localized at the apicalmost sites of lateral membranes of luminal epithelial cells in the prostate gland, whereas claudin-1 and -7 were distributed along the basolateral membranes of the epithelium. These findings provide basic information for elucidating the significance of claudins in prostate diseases, including prostate cancers.

Gap junctions are considered to play a crucial role in differentiation of epithelial cells and to be associated with tight junction proteins. In this study, to investigate the role of gap junctions in regulation of the barrier function and fence function on the tight junctions, we introduced the Cx26 gene into human airway epithelial cell line Clau-3 and used a disruption model of tight junctions employing the Na(+)/K(+)-ATPase inhibitor ouabain. In parental Calu-3 cells, gap junction proteins Cx32 and Cx43, but not Cx26, and tight junction proteins occludin, JAM-1, ZO-1, claudin-1, -2, -3, -4, -5, -6, -7, -8, -9, and -14 were detected by RT-PCR. The barrier function and fence function of tight junctions were well maintained, whereas the GJIC was low level. Treatment with ouabain caused disruption of the barrier function and fence function of tight junctions together with down-regulation of occludin, JAM-1, claudin-2, and -4 and up-regulation of ZO-1 and claudin-14. In Cx26 transfectants, Cx26 protein was detected by Western blotting and immunocytochemistry, and many gap junction plaques were observed with well-developed tight junction strands. Expression of claudin-14 was significantly increased in Cx26 transfectants compared to parental cells, and in some cells, Cx26 was co-localized with claudin-14. Interestingly, transfection with Cx26 prevented disruption of both functions of tight junctions by treatment with ouabain without changes in the tight junction proteins. Pretreatment with the GJIC blockers 18beta-glycyrrhetinic acid and oleamide did not affect the changes induced by Cx26 transfection. These results suggest that Cx26 expression, but not the mediated intercellular communication, may regulate tight junction barrier and fence functions in human airway epithelial cell line Calu-3.

Among tight-junction proteins, claudins, which play a key role in paracellular transport across epithelia, claudins 1 to 5 are expressed in the intestine, and changes in their abundance and/or distribution are considered to contribute to various gastrointestinal diseases. We investigated, by reverse transcription-PCR, immunoblot, and immunofluorescence analyses, which other claudin species were expressed in the mouse intestine, and whether they showed unique expression profiles. Rabbit polyclonal antibodies against mouse claudin-8, claudin-12, and claudin-15 were generated, and their specificity was verified by immunoblotting using COS-7 cells transfected with individual claudin cDNAs. Claudin-7, -8, -12, -13, and -15 appeared to be expressed in the duodenum, jejunum, ileum, and/or colon with remarkable variations in the expression levels along the intestinal tract, and had distinct subcellular localization in the intestinal epithelium. In addition, claudin-13 and -15 exhibited gradients along the crypt-surface axis of the colon. By contrast, claudin-6, -9, -10, -11, -14, -16, -18, and -19 were not observed in the intestine. Our results indicate that five additional species of claudins have very complex expression patterns along and within the intestine, and that this may reflect differences in paracellular permeable properties, providing valuable resources for studying the significance of these claudins in gastrointestinal disorders. This manuscript contains online supplemental material available at http://www.jhc.org. Please visit this article online to view these materials.

Gap-junction plaques are often observed with tight-junction strands of vascular endothelial cells but the molecular interaction and functional relationships between these two junctions remain obscure. We herein show that gap-junction proteins connexin40 (Cx40) and Cx43 are colocalized and coprecipitated with tight-junction molecules occludin, claudin-5, and ZO-1 in porcine blood-brain barrier (BBB) endothelial cells. Gap junction blockers 18beta-glycyrrhetinic acid (18beta-GA) and oleamide (OA) did not influence expression of Cx40, Cx43, occludin, claudin-5, junctional adhesion molecule (JAM)-A, JAM-B, JAM-C, or ZO-1, or their subcellular localization in the porcine BBB endothelial cells. In contrast, these gap-junction blocking agents inhibited the barrier function of tight junctions in cells, determined by measurement of transendothelial electrical resistance and paracellular flux of mannitol and inulin. 18beta-GA also significantly reduced the barrier property in rat lung endothelial (RLE) cells expressing doxycycline-induced claudin-1, but did not change the interaction between Cx43 and either claudin-1 or ZO-1, nor their expression levels or subcellular distribution. These findings suggest that Cx40- and/or Cx43-based gap junctions might be required to maintain the endothelial barrier function without altering the expression and localization of the tight-junction components analyzed., (Copyright 2006 Wiley-Liss, Inc.)

We investigated the mechanism by which a GnRH agonist (GnRHa) affects ovarian vascularity, vascular permeability, and expression of the tight junction protein claudin-5 in a rat model of ovarian hyperstimulation syndrome (OHSS). Hyperstimulated rats received excessive doses of pregnant mare serum gonadotropin (PMSG; 50 IU/d) for 4 consecutive days, from d 25 to 28 of life, followed by 25 IU human chorionic gonadotropin (hCG) on d 29. Control rats received 10 IU PMSG on d 27 of life, followed by 10 IU hCG on d 29. GnRHa (leuprolide 100 microg/kg.d) was administered to some hyperstimulated rats either on d 29 and 30 (short-term GnRHa treatment) or from d 25 to 30 (long-term GnRHa treatment). Ovarian vascular density (vessels per 10 mm(2)) and vessel endothelial area (percent) were assessed by immunohistochemical analysis of the distribution of von Willebrand factor, whereas vascular permeability was evaluated based on leakage of Evans blue. High doses of PMSG and hCG significantly increased ovarian weight, vascular permeability, vascular density, and the vessel endothelial area and significantly reduced expression of claudin-5 protein and mRNA. All of these effects were significantly and dose-dependently inhibited by administration of GnRHa. This suggests that reduced expression of claudin-5 plays a crucial role in the increased ovarian vascular permeability seen in OHSS and that its expression can be modulated by GnRHa treatment. Indeed, preventing redistribution of tight junction proteins in endothelial cells and the resultant loss of endothelial barrier architecture might be the key to protecting patients against massive extravascular fluid accumulation in cases of OHSS.

Disruption of the epithelial barrier function of tight junctions by the proinflammatory cytokine interferon (IFN)-gamma plays a fundamental role in the pathogenesis of inflammatory bowel disease and other gastrointestinal disorders, but its precise mechanism has not been established. This Perspective provides an overview of how IFN-gamma triggers barrier dysfunction and bacterial translocation in intestinal epithelial cells, and highlights the roles of internalization of tight-junction transmembrane proteins, as well as rearrangements of the cortical actin cytoskeleton, in intestinal inflammation.

We previously reported that expression of tight-junction molecules occludin, claudin-6 and claudin-7, as well as establishment of epithelial polarity, was triggered in mouse F9 cells expressing hepatocyte nuclear factor (HNF)-4alpha [H. Chiba, T. Gotoh, T. Kojima, S. Satohisa, K. Kikuchi, M. Osanai, N. Sawada. Hepatocyte nuclear factor (HNF)-4alpha triggers formation of functional tight junctions and establishment of polarized epithelial morphology in F9 embryonal carcinoma cells, Exp. Cell Res. 286 (2003) 288-297]. Using these cells, we examined in the present study behavior of tight-junction, adherens-junction and cell polarity proteins and elucidated the molecular mechanism behind HNF-4alpha-initiated junction formation and epithelial polarization. We herein show that not only ZO-1 and ZO-2, but also ZO-3, junctional adhesion molecule (JAM)-B, JAM-C and cell polarity proteins PAR-3, PAR-6 and atypical protein kinase C (aPKC) accumulate at primordial adherens junctions in undifferentiated F9 cells. In contrast, CRB3, Pals1 and PATJ appeared to exhibit distinct subcellular localization in immature cells. Induced expression of HNF-4alpha led to translocation of these tight-junction and cell polarity proteins to beltlike tight junctions, where occludin, claudin-6 and claudin-7 were assembled, in differentiated cells. Interestingly, PAR-6, aPKC, CRB3 and Pals1, but not PAR-3 or PATJ, were also concentrated on the apical membranes in differentiated cells. These findings indicate that HNF-4alpha provokes not only expression of tight-junction adhesion molecules, but also modulation of subcellular distribution of junction and cell polarity proteins, resulting in junction formation and epithelial polarization.

The tight junction (TJ) regulates epithelial cell polarity and barrier including permeability of the paracellular pathway. Occludin was the first integral membrane protein to be discovered, but it is not indispensable for the formation of TJ strands. The physiological function of occludin is still unclear, although occludin-deficient mice show very complex abnormalities in various organs without overt dysfunction of the TJ. To investigate the role of occludin in TJ expression and apoptosis regulated by survival signal transduction pathways such as MAPK and Akt, we performed primary culture of hepatocytes and established hepatic cell lines from occludin-deficient mice. In primary cultures of occludin-deficient mouse hepatocytes, claudin-2 expression and apoptosis were induced by down-regulation of the activation of MAPK and Akt. In the hepatic cell lines derived from occludin-deficient mice, claudin-2 expression and serum-free induced apoptosis were also increased by down-regulation of the activation of MAPK and Akt. Furthermore, in the hepatic cell lines transiently transfected with mouse and rat occludin genes, induction of claudin-2 expression and the apoptosis were inhibited with increases in activation of MAPK and Akt. These findings show that occludin plays a crucial role in claudin-2-dependent TJ function and the apoptosis involving MAPK and Akt signaling pathways in hepatocytes.

Background/aims: Hepatocyte regeneration is considered to be associated with adaptive changes in expression of gap and tight junctions through multiple signal transduction pathways including p38 MAP-kinase. The role of the stress responsitive MAP-kinase, p38 MAP-kinase, signaling pathway in function of gap and tight junctions was examined during regeneration of rat hepatocytes in vivo and in vitro., Methods: We examined changes in formation, expression and function of gap and tight junctions in rat livers after 70% partial hepatectomy and in primary cultures of rat hepatocytes, by using a p38 MAP-kinase inhibitor, SB203580., Results: When p38 MAP-kinase was activated during partial hepatectomy, down-regulation of Cx32 and up-regulation of claudin-1 were observed. By SB203580 treatment, the down-regulation of Cx32 was inhibited and the up-regulation of claudin-1 was enhanced, well maintaining the structures of gap and tight junctions. SB203580 treatment did not affect the increase of hepatocyte proliferation. In EGF induced proliferative rat hepatocytes treated with SB203580, the expression and function of Cx32 and claudin-1 were increased., Conclusions: Dynamic changes of formation of gap and tight junctions during regeneration of rat hepatocytes in vivo and in vitro are in part controlled via a p38 MAP-kinase signaling pathway, and are independent of cell growth.

The epithelial barrier of the upper respiratory tract plays a crucial role in host defense. In this study, to elucidate whether there is antigen monitoring by dendritic cells (DCs) beyond the epithelial tight-junction barrier in allergic rhinitis, we investigated the expression and function of tight junctions and characterized DCs in the epithelium of nasal mucosa from patients with allergic rhinitis. In reverse transcription-polymerase chain reaction, mRNAs of tight-junction proteins occludin, JAM-1, ZO-1, and claudin-1, -4, -7, -8, -12, -13, and -14 were detected in the nasal mucosa. Occludin, JAM-1, and ZO-1 were colocalized in the uppermost layer in the pseudostratified epithelium of the nasal mucosa, whereas claudin-1, -4, and -7 were found throughout the epithelium. In freeze-fracture replicas of the nasal mucosa, continuous tight-junction strands formed well-developed networks. Epithelial barrier function measured by a dye tracer was well maintained in occludin-positive tight junctions in the epithelium of the nasal mucosa. HLA-DR- and CD11c-positive DCs expressed claudin-1 and penetrated beyond occludin in the epithelium of the nasal mucosa with, but not without, allergic rhinitis. These results indicate that DCs may easily access antigens beyond epithelial tight junctions in the human nasal mucosa of allergic rhinitis.

Gap junctions are considered to play a crucial role in differentiation of epithelial cells, including hepatocytes. Recently, we found that Cx32 but not Cx26 was closely related to tight junctional proteins in primary cultured rat hepatocytes (Kojima et al., Exp Cell Res 263:193-201, 2001) and that Cx32 formation and/or Cx32-mediated intercellular communication could induce expression and function of tight junctions in a mouse hepatic cell line (Kojima et al., Exp Cell Res 276:40-51, 2002). In this study, to investigate the mechanisms of induction of tight junctions by transfection with Cx32, we performed cDNA microarray analysis of Cx32 transfectants, compared with parental cells derived from Cx32-deficient hepatocytes. In cDNA microarray analysis, a 2.5-fold increase in expression of membrane-associated guanylate kinase with inverted orientation-1 (MAGI-1), which is known to be localized at adherens and tight junction regions, was observed. High expression of MAGI-1 in Cx32 transfectants was confirmed by Western blotting and RT-PCR. MAGI-1 was colocalized with occludin, claudin-2, ZO-1, and F-actin, but not with E-cadherin in the apical-most regions at cell borders of Cx32 transfectants, similar to junctional adhesion molecule-1 (JAM-1), which may play a crucial role in formation and assembly of tight junctions. Treatment with the gap junction blocker 18beta-glycyrrhetinic acid did not affect expression of MAGI-1 and JAM-1 in Cx32 transfectants. These results suggest that Cx32 expression is in part related to induction of tight junctions through modulation of MAGI-1 expression in an immortalized mouse hepatic cell line.

The human palatine tonsils have surface and crypt stratified epithelium and may be initiated via the epithelium to mount immune responses to various presenting antigens. Here we investigated the expression and function of tight junctions in the epithelium of human palatine tonsils from patients with tonsillar hypertrophy or recurrent tonsillitis. Occludin, ZO-1, JAM-1, and claudin-1, -3, -4, -7, -8, and -14 mRNAs were detected in tonsillar hypertrophy. Occludin and claudin-14 were expressed in the uppermost layer of the tonsil surface epithelium, whereas ZO-1, JAM-1, and claudin-1, -4, and -7 were found throughout the epithelium. In the crypt epithelium, claudin-4 was preferentially expressed in the upper layers. In freeze-fracture replicas, short fragments of continuous tight junction strands were observed but never formed networks. In the crypt epithelium of recurrent tonsillitis, the tracer was leaked from the surface regions where occludin and claudin-4 disappeared. Occludin, ZO-1, JAM-1, and claudin-1, -3, -4, and -14, but not claudin-7, mRNAs were decreased in recurrent tonsillitis compared with those of tonsillar hypertrophy. These studies suggest unique expression of tight junctions in human palatine tonsillar epithelium, and the crypt epithelium may possess an epithelial barrier different from that of the surface epithelium.

We have recently shown that cyclic AMP (cAMP) increases claudin-5 immunoreactivity along cell boundaries and could promote phosphorylation of claudin-5 on threonine residues in porcine blood-brain barrier (BBB) endothelial cells via a protein kinase A (PKA)-dependent pathway (Exp. Cell Res. 290 [2003] 275). Along this line, we identified a putative phosphorylation site for PKA at Thr(207) in the intracytoplasmic carboxyl terminal domain of claudin-5. To clarify the biological significance of this site in regulation of endothelial barrier functions, we established rat lung endothelial (RLE) cells expressing doxycycline (Dox)-inducible wild-type claudin-5 and a mutant with a substitution of Ala for Thr(207) (CL5T207A). We show that induction of wild-type claudin-5 is sufficient to reconstitute the paracellular barrier against inulin (5 kDa), but not mannitol (182 Da), in leaky RLE cells. By contrast, the barrier against both molecules was induced in the mutant cells. We also demonstrate that, upon cAMP treatment, Thr(207) of claudin-5 is involved in enhancement of claudin-5 immunoreactive signals along cell borders, rapid reduction in transendothelial electrical resistance (TER), and loosening of the claudin-5-based endothelial barrier against mannitol, but not inulin. cAMP decreased the claudin-5-based endothelial barrier, strongly suggesting that other tight-junction molecule(s) are required to elevate endothelial barrier functions in response to cAMP.

Mitogen-activated protein kinase (MAPK) modulates the barrier function of tight junctions. We identified a putative phosphorylation site for MAPK at around Thr203 (PKPTP) in claudin-1, and determined the biological significance of this site. To this end, using the rat lung endothelial cell line RLE, we generated cells expressing doxycycline (Dox)-inducible wild-type claudin-1 and its mutant with substitution of Thr203 to Ala, and named them RLE:rtTA:CL1 and RLE:rtTA:CL1T203A, respectively. We herein show, by measurement of transendothelial electrical resistance and paracellular flux of mannitol and inulin, that functional tight junctions were reconstituted in both cells by Dox-induced expression of claudin-1. Interestingly, the barrier functions of tight junctions were less developed in RLE:rtTA:CL1T203A cells compared with RLE:rtTA:CL1 cells. Consistently, levels of both detergent-insoluble claudin-1 protein and its threonine-phosphorylation after Dox treatment were low in RLE:rtTA:CL1T203A cells compared to RLE:rtTA:CL1 cells. Furthermore, pretreatment with the MAPK inhibitor PD98059 markedly suppressed the barrier function and amount of detergent-insoluble claudin-1 in Dox-exposed RLE:rtTA:CL1 cells, whereas it marginally influenced those in RLE:rtTA:CL1T203A cells. These findings indicate that Thr203 of claudin-1 is required to enhance the barrier function of claudin-1-based tight junctions, probably via its phosphorylation and subsequent integration into tight junctions.

The tight junctions of the glandular epithelium are crucial for the maintenance of cell polarity, separating the plasma membrane into apical and basolateral domains. Thus abnormalities of the tight junctions may result in the structural disturbances of glandular epithelial neoplasia. In this study we introduced an anti-occludin monoclonal antibody for semiquantitative assay of the occludin expression in tissue sections of human normal and neoplastic endometrial epithelia using the Adobe Photoshop and NIH Image programs. Normal endometrial glands and samples of endometrial hyperplasia and endometrioid carcinoma grade 1 fully expressed occludin at the apical cell border. In endometrioid carcinomas grades 2 and 3, however, occludin disappeared in solid areas of the carcinomatous tissues. Occludin was also found at the apical borders of the cancer cells that formed glandular structures. Occludin expression decreased progressively in parallel with the increase in carcinoma grade, and the decreased occludin expression correlated with myometrial invasion and lymph node metastasis. These results suggest that the loss of tight junctions has a close relationship with structural atypia in the progression of human endometrial carcinomas and their malignant potential.

Hepatocytes tightly connect with each other by intercellular junctions to form liver cell plates. The junctions composed of gap, tight, and adherens junctions and desmosomes concentrate around bile canaliculi. In particular, tight junctions serve as a barrier to keep bile in bile canaliculi away from the blood circulation. Thus, it is very reasonable to call tight junctions of hepatocytes the blood-biliary barrier. On the other hand, gap junctions of hepatocytes are considered to enable ordered contraction of bile canaculi from centrizonal to periportal hepatocytes by their function of intercellular communication. Gap and tight junctions may thus play a crucial role in bile secretion, one of the most differentiated functions of the liver. In intrahepatic cholestasis, a common pathological condition of the liver, downregulation of gap and tight junctional functions is seen, which results in impaired intercellular communication and in leaky tight junctions. Although the changes in gap and tight junctions had been considered to be independent of each other, recent findings that the tight junction-associated proteins ZO-1 and occludin bind to connexins indicate the possibility of either coordinate or reciprocal regulation of macromolecular complexes containing gap- and tight-junction proteins. In this review, we introduce the interaction and regulation between gap and tight junctions of hepatocytes in vitro and discuss the regulatory mechanisms of the "blood-biliary barrier" to study the molecular pathogenesis of cholestasis.

Tight junctions are intercellular junctions adjacent to the apical end of the lateral membrane surface. They have two functions, the barrier (or gate) function and the fence function. The barrier function of tight junctions regulates the passage of ions, water, and various macromolecules, even of cancer cells, through paracellular spaces. The barrier function is thus relevant to edema, jaundice, diarrhea, and blood-borne metastasis. On the other hand, the fence function maintains cell polarity. In other words, tight junctions work as a fence to prevent intermixing of molecules in the apical membrane with those in the lateral membrane. This function is deeply involved in cancer cell biology, in terms of loss of cell polarity. Of the proteins comprising tight junctions, integral membrane proteins occludin, claudins, and JAMs have been recently discovered. Of these molecules, claudins are exclusively responsible for the formation of tight-junction strands and are connected with the actin cytoskeleton mediated by ZO-1. Thus, both functions of tight junctions are dependent on the integrity of the actin cytoskeleton as well as ATP. Mutations in the claudin14 and the claudin16 genes result in hereditary deafness and hereditary hypomagnesemia, respectively. Some pathogenic bacteria and viruses target and affect the tight-junction function, leading to diseases. In this review, the relationship between tight junctions and human diseases is summarized.

F9 murine embryonal carcinoma cells provide an attractive system for facilitating molecular mechanisms for epithelial morphogenesis, since they have the capability of differentiating into polarized epithelial cells bearing an apical junctional complexes. We previously showed that a specific retinoid X receptor-retinoic acid receptor heterodimer transduced retinoid signals for biogenesis of functional tight junctions in F9 cells (Exp. Cell Res. 263, (2001) 163). In the present study we generated F9 cells expressing doxycycline-inducible hepatocyte nuclear factor (HNF)-4alpha, a nuclear receptor. We herein show that induction of HNF-4alpha initiates differentiation of F9 cells to polarized epithelial cells, in which tight-junction proteins occludin, claudin-6, claudin-7, and ZO-1 are concentrated at the apical-most regions of lateral membranes. Expression of occludin, claudin-6, and claudin-7 was induced in the cells by doxycycline treatment in a dose- and time-dependent manner, in terms of the amount of HNF-4alpha. In contrast, expression levels of ZO-1, ZO-2, E-cadherin, and beta-catenin were not altered by HNF-4alpha. We also demonstrate, by analysis of diffusion of labeled sphingomyelin, that the fence function of tight junctions is achieved by induction of HNF-4alpha. These findings indicate that HNF-4alpha triggers de novo formation of functional tight junctions and establishment of epithelial cell polarity.

This study we presents a novel anti-occludin monoclonal antibody that can be used for formalin-fixed, paraffin-embedded tissue sections. The relationships between aberrant localization of carcinoembryonic antigen (CEA) and abnormalities of tight junctions were studied in human colorectal cancers by this antibody. Abnormalities in the cell surface expression of CEA have been shown to be characteristic of human colorectal cancer cells. Cancer cells that participated in the formation of glandular structures expressed occludin at the apical cell border and CEA was expressed more apically than occludin. Where cancer cells showed solid nests without glandular structures, occludin was completely lost and CEA was demonstrated in a diffuse pattern throughout the cells. These findings suggest that the polarized apical expression of CEA in neoplastic glandular structures depends on the expression of occludin and the fence function of tight junctions. During tumour progression, loss of occludin may lead to the loss of membrane polarity and the non-polarized expression of CEA. The antibody described provides a powerful tool for the study of tight junctions in surgically resected human tissue., (Copyright 2002 John Wiley & Sons, Ltd.)

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.