Connexin 26 expression prevents down-regulation of barrier and fence functions of tight junctions by Na+/K+-ATPase inhibitor ouabain in human airway epithelial cell line Calu-3

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.     

Epidermal growth factor modulates claudins and tight junctional functions in ovarian cancer cell lines

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.     

Protein kinase Cα inhibitor enhances the sensitivity of human pancreatic cancer HPAC cells to <Emphasis Type="Italic">Clostridium perfringens</Emphasis> enterotoxin via claudin-4

Protein kinase C (PKC) is overexpressed in cancer, including pancreatic cancer, compared with normal tissue. Moreover, PKCα is considered one of the biomarkers for the diagnosis of cancers. In several human cancers, the claudin tight junction molecules are abnormally regulated and are thus promising molecular targets for diagnosis and therapy with Clostridium perfringens enterotoxin (CPE). In order to investigate the changes of tight junction functions of claudins via PKCα activation in pancreatic cancer cells, the well-differentiated human pancreatic cancer cell line HPAC, with its highly expressed tight junction molecules and well-developed barrier function, was treated with the PKC activator 12-O-tetradecanoylphorbol 13-acetate (TPA). Treatment with TPA modified the activity of phosphoPKCα and caused an increase of the Snail family members Snail, Slug and Smad-interacting protein 1 and a decrease of E-cadherin. In HPAC cells treated with TPA, downregulation of claudin-1 and mislocalization of claudin-4 and occludin around the nuclei were observed, together with a decrease in the numbers of tight junction strands and an increase in phosphorylation of claudin-4. The barrier function and the cytotoxicity of CPE were significantly decreased on TPA treatment. All such changes after TPA treatment were prevented by inhibitors of panPKC and PKCα. These findings suggest that, in human pancreatic cancer cells, PKCα activation downregulates tight junction functions as a barrier and as a receptor of CPE via the modification of claudin-1 and −4 during epithelial to mesenchymal transition-like changes. PKCα inhibitors might represent potential therapeutic agents against human pancreatic cancer cells by use of CPE cytotoxicity via claudin-4.     

Clostridium perfringens enterotoxin fragment removes specific claudins from tight junction strands: Evidence for direct involvement of claudins in tight junction barrier.

Claudins, comprising a multigene family, constitute tight junction (TJ) strands. Clostridium perfringens enterotoxin (CPE), a single approximately 35-kD polypeptide, was reported to specifically bind to claudin-3/RVP1 and claudin-4/CPE-R at its COOH-terminal half. We examined the effects of the COOH-terminal half fragment of CPE (C-CPE) on TJs in L transfectants expressing claudin-1 to -4 (C1L to C4L, respectively), and in MDCK I cells expressing claudin-1 and -4. C-CPE bound to claudin-3 and -4 with high affinity, but not to claudin-1 or -2. In the presence of C-CPE, reconstituted TJ strands in C3L cells gradually disintegrated and disappeared from their cell surface. In MDCK I cells incubated with C-CPE, claudin-4 was selectively removed from TJs with its concomitant degradation. At 4 h after incubation with C-CPE, TJ strands were disintegrated, and the number of TJ strands and the complexity of their network were markedly decreased. In good agreement with the time course of these morphological changes, the TJ barrier (TER and paracellular flux) of MDCK I cells was downregulated by C-CPE in a dose-dependent manner. These findings provided evidence for the direct involvement of claudins in the barrier functions of TJs.     

Thymic stromal lymphopoietin induces tight junction protein claudin-7 via NF-κB in dendritic cells

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-κB 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-κB as well as via TLRs and may control tight junctions of DCs to preserve the epithelial barrier during allergic inflammation.     

Clostridium perfringens enterotoxin binds to the second extracellular loop of claudin-3, a tight junction integral membrane protein

Claudins (claudin-1 to -18) with four transmembrane domains and two extracellular loops constitute tight junction strands. The peptide toxin Clostridium perfringens enterotoxin (CPE) has been shown to bind to claudin-3 and -4, but not to claudin-1 or -2. We constructed claudin-1/claudin-3 chimeric molecules and found that the second extracellular loop of claudin-3 conferred CPE sensitivity on L fibroblasts. Furthermore, overlay analyses revealed that the second extracellular loop of claudin-3 specifically bound to CPE at the K(a) value of 1.0x10(8) M(-1). We concluded that the second extracellular loop is the site through which claudin-3 interacts with CPE on the cell surface.     

Developmental regulation of claudin localization by fetal alveolar epithelial cells

Tight junction proteins in the claudin family regulate epithelial barrier function. We examined claudin expression by human fetal lung (HFL) alveolar epithelial cells cultured in medium containing dexamethasone, 8-bromo-cAMP, and isobutylmethylxanthanine (DCI), which promotes alveolar epithelial cell differentiation to a type II phenotype. At the protein level, HFL cells expressed claudin-1, claudin-3, claudin-4, claudin-5, claudin-7, and claudin-18, where levels of expression varied with culture conditions. DCI-treated differentiated HFL cells cultured on permeable supports formed tight transepithelial barriers, with transepithelial resistance (TER) >1,700 ohm/cm(2). In contrast, HFL cells cultured in control medium without DCI did not form tight barriers (TER <250 ohm/cm(2)). Consistent with this difference in barrier function, claudins expressed by HFL cells cultured in DCI medium were tightly localized to the plasma membrane; however, claudins expressed by HFL cells cultured in control medium accumulated in an intracellular compartment and showed discontinuities in claudin plasma membrane localization. In contrast to claudins, localization of other tight junction proteins, zonula occludens (ZO)-1, ZO-2, and occludin, was not sensitive to HFL cell phenotype. Intracellular claudins expressed by undifferentiated HFL cells were localized to a compartment containing early endosome antigen-1, and treatment of HFL cells with the endocytosis inhibitor monodansylcadaverine increased barrier function. This suggests that during differentiation to a type II cell phenotype, fetal alveolar epithelial cells use differential claudin expression and localization to the plasma membrane to help regulate tight junction permeability.     

Enhancement of barrier function by overexpression of claudin-4 in tight junctions of submandibular gland cells

In salivary glands, primary saliva is produced by acini and is modified by the reabsorption and secretion of ions in the ducts. Thus, the permeability of intercellular junctions in the ducts is considered to be lower than in the acini. We have examined the relationship between the expressed claudin isotypes and the barrier functions of tight junctions in a submandibular gland epithelial cell line, SMIE. SMIE cells were originally derived from rat submandibular duct cells, but their barrier functions are not as efficient as those of Madin-Darby canine kidney cells. Large molecules, such as 70-kDa dextran, diffuse across the monolayers, although E-cadherin and occludin, adherens junction and tight junction proteins, respectively, are expressed in SMIE cells. Claudin-3 protein has also been detected, but the expression level of claudin-3 mRNA is much lower than in the original submandibular glands. Other claudins including claudin-4 (originally expressed in the duct cells) have not been detected. Because of the limited expression of claudins, SMIE cells are suitable for studying the role(s) of claudins. To examine the function of claudin-4 in submandibular glands, we have overexpressed green fluorescence protein (GFP)-fused claudin-4 in SMIE cells. Cells that express GFP-fused claudin-4 have a higher transepithelial electrical resistance and a lower permeability of 70-kDa dextran, although the expression levels of occludin and claudin-3 are hardly affected. Therefore, claudin-4 plays a role in the regulation of the barrier function of tight junctions in submandibular glands. This work was supported by Grants-in-Aid for scientific research from the Ministry of Education, Science, Culture, Sports, and Technology of Japan (16591868), by a Nihon University Multidisciplinary Research Grant for 2006 and 2007, and by a Grant-in-Aid for a 2003 Multidisciplinary Research Project from MEXT.     

Aspirin induces gastric epithelial barrier dysfunction by activating p38 MAPK via claudin-7

Tight junctions create a paracellular permeability barrier that is breached when nonsteroidal anti-inflammatory drugs cause gastrointestinal injury, including increased gastrointestinal permeability. However, the mechanism by which aspirin affects the function of gastric epithelial tight junctions is unknown. Thus, we examined the effect of aspirin on gastric mucosal barrier properties and tight junction organization using MKN28, a human gastric epithelial cell line that expresses claudin-3, claudin-4, claudin-7, zonula occludens (ZO)-1, and occludin, but not claudin-2 or claudin-5, as determined by immunoblot analysis and immunofluorescent staining. Aspirin (5 mM) treatment of MKN28 gastric epithelial monolayers significantly decreased transepithelial electrical resistance and increased dextran permeability. Both aspirin-mediated permeability and phosphorylation of p38 MAPK were significantly attenuated by SB-203580 (a p38 MAPK inhibitor) but not by U-0126 (a MEK1 inhibitor) or SP-600125 (a JNK inhibitor). Aspirin significantly decreased the quantity of claudin-7 protein produced by MKN28 cells but not the quantity of claudin-3, claudin-4, ZO-1, or occludin. The aspirin-induced decrease in claudin-7 protein was completely abolished by SB-203580 pretreatment. These results demonstrate, for the first time, that claudin-7 protein is important in aspirin-induced gastric barrier loss and that p38 MAPK activity mediates this epithelial barrier dysfunction. tight junction; p38 mitogen-activated protein kinase; permeability     

Expression and function of tight junction associated molecules in human breast tumor cells is not affected by the Ras-MEK1 pathway.

Constitutive activation of Ras or Ras-mediated signaling pathways is one of the initial steps during tumorigenesis that promotes neoplastic transformation. Recently it was reported that in Ha-Ras overexpressing MDCK cells the tight junction proteins claudin-1, occludin and ZO-1 were absent at cell-cell contact sites but present in the cytoplasm. Inhibition of MEK1 activity recruited all three proteins to the cell membrane leading to a restoration of the tight junction barrier function in MDCK cells. In order to evaluate the relevance of the MEK1 pathway in tight junction regulation in breast cancer cells, we investigated the effect ofMEK1 inhibition on expression of claudin-1, occludin and ZO-1 in natively claudin-1 expressing T47-D cells (low Ras activity), claudin-1 negative MCF-7 cells (elevated Ras activity) as well as two retroviral claudin-1 transduced MCF-7 daughter cell lines with prominent membrane and cytoplasmic claudin-1 dominant homing, respectively. Although we effectively blocked phosphorylation of MAPKs ERK-1 and ERK-2 using the selective MEK1 inhibitor PD98059, no quantitative changes of mRNA or protein levels of claudin-1, occludin and ZO-1 could be detected in all cell lines investigated. Furthermore, immnfluorescence analysis of claudin-1 revealed that inhibition of the MAPK pathway did not alter th e subcellular cytoplasmic distribution of claudin-1 to be more membrane specific. Finally, the diffusion barrier properties of tight junctions as analyzed by transepithelial resistance (TER) or paracellular flux analysis of 3 and 40 kDa dextran of tight junctions were not altered in the claudin-1 positive T47-D and the MCF-7 cell lines. Our findings indicate that the proposed involvement of the Ras-MEK-ERK pathway is likely not involved in the dysregulated tight junction formation in breast tumor cells and indicates that elevated activity of Ras might not be of general importance for the disruption of tight junction structures in breast tumors.     

Claudin-2 forms homodimers and is a component of a high molecular weight protein complex

Tight junctions are multiprotein complexes that form the fundamental physiologic and anatomic barrier between epithelial and endothelial cells, yet little information is available about their molecular organization. To begin to understand how the transmembrane proteins of the tight junction are organized into multiprotein complexes, we used blue native-PAGE (BN-PAGE) and cross-linking techniques to identify complexes extracted from MDCK II cells and mouse liver. In nonionic detergent extracts from MDCK II cells, the tight junction integral membrane protein claudin-2 was preferentially isolated as a homodimer, whereas claudin-4 was monomeric. Analysis of the interactions between chimeras of claudin-2 and -4 are consistent with the transmembrane domains of claudin-2 being responsible for dimerization, and mutational analysis followed by cross-linking indicated that the second transmembrane domains were arranged in close proximity in homodimers. BN-PAGE of mouse liver membrane identified a relatively discrete high molecular weight complex containing at least claudin-1, claudin-2, and occludin; the difference in the protein complex sizes between cultured cells and tissues may reflect differences in tight junction protein or lipid composition or post-translational modifications. Our results suggest that BN-PAGE may be a useful tool in understanding tight junction structure.     

Claudin-3 acts as a sealing component of the tight junction for ions of either charge and uncharged solutes

The paracellular barrier of epithelia and endothelia is established by several tight junction proteins including claudin-3. Although claudin-3 is present in many epithelia including skin, lung, kidney, and intestine and in endothelia, its function is unresolved as yet. We therefore characterized claudin-3 by stable transfection of MDCK II kidney tubule cells with human claudin-3 cDNA. Two clone systems were analyzed, exhibiting high or low claudin-2 expression, respectively. Expression of other claudins was unchanged. Ultrastructurally, tight junction strands were changed toward uninterrupted and rounded meshwork loops. Functionally, the paracellular resistance of claudin-3-transfected monolayers was strongly elevated, causing an increase in transepithelial resistance compared to vector controls. Permeabilities for mono- and divalent cations and for anions were decreased. In the high-claudin-2 system, claudin-3 reduced claudin-2-induced cation selectivity, while in the low-claudin-2 system no charge preference was observed, the latter thus reflecting the "intrinsic" action of claudin-3. Furthermore, the passage of the paracellular tracers fluorescein (332 Da) and FD-4 (4 kDa) was decreased, whereas the permeability to water was not affected. We demonstrate that claudin-3 alters the tight junction meshwork and seals the paracellular pathway against the passage of small ions of either charge and uncharged solutes. Thus, in a kidney model epithelium, claudin-3 acts as a general barrier-forming protein.     

Rab14 regulation of claudin-2 trafficking modulates epithelial permeability and lumen morphogenesis

Regulation of epithelial barrier function requires targeted insertion of tight junction proteins that have distinct selectively permeable characteristics. The insertion of newly synthesized proteins and recycling of internalized tight junction components control both polarity and junction function. Here we show that the small GTPase Rab14 regulates tight junction structure. In Madin–Darby canine kidney (MDCK) II cells, Rab14 colocalizes with junctional proteins, and knockdown of Rab14 results in increased transepithelial resistance. In cells without Rab14, there are small changes in the trafficking of claudin-1 and occludin. In addition, there is substantial depletion of the leaky claudin, claudin-2, but not other tight junction components. The loss of claudin-2 is complemented by inhibition of lysosomal function, suggesting that Rab14 sorts claudin-2 out of the lysosome-directed pathway. MDCK I cells lack claudin-2 endogenously, and knockdown of Rab14 in these cells does not result in a change in transepithelial resistance, suggesting that the effect is specific to claudin-2 trafficking. Furthermore, leaky claudins have been shown to be required for epithelial morphogenesis, and knockdown of Rab14 results in failure to form normal single-lumen cysts in three-dimensional culture. These results implicate Rab14 in specialized trafficking of claudin-2 from the recycling endosome.     

Rapid disruption of intestinal barrier function by gliadin involves altered expression of apical junctional proteins

Coeliac disease is a chronic enteropathy caused by the ingestion of wheat gliadin and other cereal prolamines derived from rye and barley. In the present work, we investigated the mechanisms underlying altered barrier function properties exerted by gliadin-derived peptides in human Caco-2 intestinal epithelial cells. We demonstrate that gliadin alters barrier function almost immediately by decreasing transepithelial resistance and increasing permeability to small molecules (4 kDa). Gliadin caused a reorganisation of actin filaments and altered expression of the tight junction proteins occludin, claudin-3 and claudin-4, the TJ-associated protein ZO-1 and the adherens junction protein E-cadherin.     

The PIKfyve inhibitor YM201636 blocks the continuous recycling of the tight junction proteins claudin-1 and claudin-2 in MDCK cells

Tight junctions mediate the intercellular diffusion barrier found in epithelial tissues but they are not static complexes; instead there is rapid movement of individual proteins within the junctions. In addition some tight junction proteins are continuously being endocytosed and recycled back to the plasma membrane. Understanding the dynamic behaviour of tight junctions is important as they are altered in a range of pathological conditions including cancer and inflammatory bowel disease. In this study we investigate the effect of treating epithelial cells with a small molecule inhibitor (YM201636) of the lipid kinase PIKfyve, a protein which is involved in endocytic trafficking. We show that MDCK cells treated with YM201636 accumulate the tight junction protein claudin-1 intracellularly. In contrast YM201636 did not alter the localization of other junction proteins including ZO-1, occludin and E-cadherin. A biochemical trafficking assay was used to show that YM201636 inhibited the endocytic recycling of claudin-1, providing an explanation for the intracellular accumulation. Claudin-2 was also found to constantly recycle in confluent MDCK cells and treatment with YM201636 blocked this recycling and caused accumulation of intracellular claudin-2. However, claudin-4 showed negligible endocytosis and no detectable intracellular accumulation occurred following treatment with YM201636, suggesting that not all claudins show the same rate of endocytic trafficking. Finally, we show that, consistent with the defects in claudin trafficking, incubation with YM201636 delayed formation of the epithelial permeability barrier. Therefore, YM201636 treatment blocks the continuous recycling of claudin-1/claudin-2 and delays epithelial barrier formation.     

Altered expression of tight junction molecules in alveolar septa in lung injury and fibrosis

The dysfunction of alveolar barriers is a critical factor in the development of lung injury and subsequent fibrosis, but the underlying molecular mechanisms remain poorly understood. To clarify the pathogenic roles of tight junctions in lung injury and fibrosis, we examined the altered expression of claudins, the major components of tight junctions, in the lungs of disease models with pulmonary fibrosis. Among the 24 known claudins, claudin-1, claudin-3, claudin-4, claudin-7, and claudin-10 were identified as components of airway tight junctions. Claudin-5 and claudin-18 were identified as components of alveolar tight junctions and were expressed in endothelial and alveolar epithelial cells, respectively. In experimental bleomycin-induced lung injury, the levels of mRNA encoding tight junction proteins were reduced, particularly those of claudin-18. The integrity of the epithelial tight junctions was disturbed in the fibrotic lesions 14 days after the intraperitoneal instillation of bleomycin. These results suggest that bleomycin mainly injured alveolar epithelial cells and impaired alveolar barrier function. In addition, we analyzed the influence of transforming growth factor-β (TGF-β), a critical mediator of pulmonary fibrosis that is upregulated after bleomycin-induced lung injury, on tight junctions in vitro. The addition of TGF-β decreased the expression of claudin-5 in human umbilical vein endothelial cells and disrupted the tight junctions of epithelial cells (A549). These results suggest that bleomycin-induced lung injury causes pathogenic alterations in tight junctions and that such alterations seem to be induced by TGF-β.     

Proinflammatory cytokines tumor necrosis factor-α and interferon-γ modulate epithelial barrier function in Madin-Darby canine kidney cells through mitogen activated protein kinase signaling

Background  

The tight junction is a dynamic structure that is regulated by a number of cellular signaling processes. Occludin, claudin-1, claudin-2 and claudin-3 are integral membrane proteins found in the tight junction of MDCK cells. These proteins are restricted to this region of the membrane by a complex array of intracellular proteins which are tethered to the cytoskeleton. Alteration of these tight junction protein complexes during pathological events leads to impaired epithelial barrier function that perturbs water and electrolyte homeostasis. We examined MDCK cell barrier function in response to challenge by the proinflammatory cytokines tumor necrosis factor-α (TNFα) and interferon-γ (IFNγ).     

Suppression of the transformed phenotype of breast cancer by tropomyosin-1

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.     

Occludin S408 phosphorylation regulates tight junction protein interactions and barrier function

Although the C-terminal cytoplasmic tail of the tight junction protein occludin is heavily phosphorylated, the functional impact of most individual sites is undefined. Here, we show that inhibition of CK2-mediated occludin S408 phosphorylation elevates transepithelial resistance by reducing paracellular cation flux. This regulation requires occludin, claudin-1, claudin-2, and ZO-1. S408 dephosphorylation reduces occludin exchange, but increases exchange of ZO-1, claudin-1, and claudin-2, thereby causing the mobile fractions of these proteins to converge. Claudin-4 exchange is not affected. ZO-1 domains that mediate interactions with occludin and claudins are required for increases in claudin-2 exchange, suggesting assembly of a phosphorylation-sensitive protein complex. Consistent with this, binding of claudin-1 and claudin-2, but not claudin-4, to S408A occludin tail is increased relative to S408D. Finally, CK2 inhibition reversed IL-13-induced, claudin-2-dependent barrier loss. Thus, occludin S408 dephosphorylation regulates paracellular permeability by remodeling tight junction protein dynamic behavior and intermolecular interactions between occludin, ZO-1, and select claudins, and may have therapeutic potential in inflammation-associated barrier dysfunction.