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Tight junctions (TJs) are the most apical components of junctional complexes in epithelial and endothelial cells. Barrier function is one of the major functions of TJ, which restricts the ions and small water-soluble molecules from passing through the paracellular pathway. Adherens junctions (AJs) play an important role in cell-cell adhesion and cell signaling. Gap junctions (GJs) are intercellular channels regulating electrical and metabolic signals between cells. It is well known that TJ integral membrane proteins, such as claudins and occludins, are the molecular building blocks responsible for TJ barrier function. However, recent studies demonstrate that proteins of other junctional complexes can influence and regulate TJ barrier function. Therefore, the crosstalk between different cell junctions represents a common means to modulate cellular activities. In this review, we will discuss the interactions among TJ, AJ, and GJ by focusing on how AJ and GJ proteins regulate TJ barrier function in different biological systems.

Background: Necrotizing enterocolitis (NEC) is a leading cause of death in preterm infants. Neonates weighing <1500 grams are at the highest risk for acquiring NEC, with a prevalence of nearly 7-10%, mortality up to 30%, and several long-term complications among survivors. Despite advancements in neonatal medicine, this disease remains a challenge to treat. The aim of this study is to investigate the effect of NEC on gut epithelial tight junctions and its barrier function using a NEC mouse model., Methods: Three-day old C57BL/6 mouse pups were fed with Esbilac formula every 3 hours and then subjected to hypoxia twice daily followed by cold stress. Dam fed pups from the same litters served as controls. Pups were observed and sacrificed 96 hours after the treatments and intestines were removed for experiments. The successful induction of NEC was confirmed by histopathology. Changes in tight junction proteins in NEC intestines were studied by western blotting and immunofluorescent microscopy using specific protein markers. The gut leakage in NEC was visualized using biotin tracer molecules., Results: Our study results demonstrate that we induced NEC in >50% of experimental pups, pups lost nearly 40% of weight and their intestines showed gross changes and microscopic changes associated with NEC. There were inflammatory changes with loss of tight junction barrier function and disruption of tight junction claudin proteins in the intestines of NEC mouse model. We have demonstrated for the first time that NEC intestines develop increased leakiness as visualized by biotin tracer leakage., Conclusions: NEC leads to breakdown of epithelial barrier due to changes in tight junction proteins with increased leakiness which may explain the transmigration of microbes and microbial products from the gut lumen into the blood stream leading to sepsis like signs clinically witnessed.

Background: Necrotizing enterocolitis (NEC) is a devastating condition affecting premature infants and leads to high mortality and chronic morbidity. Severe form of NEC is associated with acute renal failure, fluid imbalance, hyponatremia, and acidosis. We investigated the effect of NEC on tight junction (TJ) proteins in kidneys using a NEC mouse model to investigate the basis for the observed renal dysfunction., Methods: NEC was induced in C57BL/6 mice by formula feeding and subjecting them to periods of hypoxia and cold stress. NEC was confirmed by gross and histological examination. We studied various markers of inflammation in kidneys and investigated changes in expression of several TJ proteins and AQP2 using immunofluorecent staining and western blotting., Results: We found markedly increased expression of NFκB, TGFβ, and ERK1/2 along with claudin-1, -2, -3, -4, -8, and AQP-2 in NEC kidneys. The membrane localization of claudin-2 was altered in the NEC kidneys and its immunostaining signal at TJ was disrupted., Conclusion: NEC led to a severe inflammatory response not only in the gut but also in the kidneys. NEC increased expression of several TJ proteins and caused disruption of claudin-2 in renal tubules. These observed changes can help explain some of the clinical findings observed in NEC.

Background & Aims: Integrity of the intestinal epithelium is required for nutrition absorption and defense against pathogens. Claudins are cell adhesion molecules that localize at tight junctions (TJs); many are expressed in the intestinal tract, but little is known about their functions. Claudin-7 is unique in that it has a stronger basolateral membrane distribution than other claudins, which localize primarily to apical TJs in the intestinal epithelium. We investigated the basolateral functions of claudin-7 and assessed the effects of disruption of Cldn7 in intestines of mice., Methods: We generated Cldn7(-/-) mice and examined their intestines by histology, molecular and cellular biology, and biochemistry approaches. We performed gene silencing experiments in epithelial cell lines using small interfering RNAs (siRNAs)., Results: The Cldn7(-/-) mice had severe intestinal defects that included mucosal ulcerations, epithelial cell sloughing, and inflammation. Intestines of Cldn7(-/-) mice produced significantly higher levels of cytokines, the nuclear factor κB p65 subunit, and cyclooxygenase 2; they also up-regulated expression of matrix metalloproteinases (MMPs)-3 and -7. siRNA in epithelial cell lines showed that the increased expression of MMP-3 resulted directly from claudin-7 depletion, whereas that of MMP-7 resulted from inflammation. Electron microscopy analysis showed that intestines of Cldn7(-/-) mice had intercellular gaps below TJs and cell matrix loosening. Deletion of Cldn7 reduced expression and altered localization of the integrin α2 subunit in addition to disrupting formation of complexes of claudin-7, integrin α2, and claudin-1 that normally form in epithelial basolateral compartments of intestines., Conclusions: In mice, claudin-7 has non-TJ functions, including maintenance of epithelial cell-matrix interactions and intestinal homeostasis., (Copyright © 2012 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Tight junctions (TJs) are the most apical component of the junctional complexes in mammalian epithelial cells and form selective paracellular barriers restricting the passage of solutes and ions across the epithelial sheets. Claudins, a TJ integral membrane protein family, play a critical role in regulating paracellular barrier permeability. In the in vitro cell culture system, transepithelial electrical resistance (TER) measurement and the flux of radioisotope or fluorescent labeled molecules with different sizes have been widely used to determine the TJ barrier function. In the in vivo system, the tracer molecule Sulfo-NHS-Biotin was initially used in Xenopus embryo system and subsequently was successfully applied to a number of animal tissues in situ and in different organisms under the experimental conditions to examine the functional integrity of TJs by several laboratories. In this chapter, we will describe the detailed procedures of applying biotin as a paracellular tracer molecule to different in vivo systems to assay TJ barrier function.

Tight junctions (TJ) constitute paracellular diffusion channels regulating the passage of ions and solutes across epithelia. We recently demonstrated that overexpression of the TJ membrane protein claudin-7 in LLC-PK1 cells decreases paracellular permeability to Cl(-) and increases paracellular permeability to Na(+). To investigate the effect of charged amino acid residues in extracellular domains (ED) of claudin-7 on paracellular charge selectivity, we created claudin-7 mutants by replacing negatively charged amino acids on ED with positively charged amino acids. Immunofluorescence light microscopy showed that these mutant proteins were correctly targeted to the cell junction. Ultrastructure examination of TJ morphology did not reveal any difference between cells expressing wildtype (WT) and mutant claudin-7. However, electrophysiological studies showed increased Cl(-) permeability in cells expressing first extracellular domain (ED1) mutants, but not second extracellular domain (ED2) mutants, compared to that of WT claudin-7. Our results demonstrate that negatively charged amino acids in ED1 of claudin-7 are involved in modulating paracellular Cl(-) permeability.

Tight junctions form the primary barrier regulating the diffusion of fluid, electrolytes and macromolecules through the paracellular pathway. Claudins are the major structural and functional components of tight junction strands and are considered as the best candidates for forming paracellular channels. They are a family of integral membrane proteins with more than 20 members and show distinct tissue distribution patterns. In this study, we found that claudin-7 is expressed in the distal and collecting tubules and the thick ascending limb of Henle of porcine and rat kidneys. To investigate the role of claudin-7 in paracellular transport, we have overexpressed a mouse claudin-7 construct in LLC-PK1 cells. Overexpression of claudin-7 did not affect the expression and localization of endogenous claudin-1, -3, -4, -7, and ZO-1. However, transepithelial electrical resistance in claudin-7-overexpressing cells was greatly increased. In addition, electrophysiological measurements revealed a dramatic reduction of dilution potentials in claudin-7-overexpressing cells compared to that of control cells. To determine which ions are responsible for the effects of claudin-7 overexpression on transepithelial electrical resistance and dilution potentials, we applied an ion substitution strategy. When NaCl was replaced with sodium aspartate, transepithelial electrical resistance was significantly decreased and dilution potentials were increased in claudin-7-overexpressing cells as compared to controls, the opposite effects from that of using NaCl. Furthermore, when NaCl was substituted by arginine-HCl or lysine-HCl, the increase in transepithelial electrical resistance was greater and the reduction in dilution potentials was smaller. Taken together, our studies demonstrated for the first time that the effect of claudin-7 overexpression in LLC-PK1 cells on paracellular transport is mediated through a concurrent decrease in the paracellular conductance to Cl(-) and an increase in the paracellular conductance to Na(+). These results support the model that claudin-7 may form a paracellular barrier to Cl(-) while acting as a paracellular channel to Na(+).

Immunoprecipitation is one of the most commonly used techniques to study protein-protein interaction in vivo. There are three major steps involved in an immunoprecipitation procedure: 1) lysis of the cells to make the antigen of interest available; 2) formation of the antibody-antigen complex by adding specific antibody; 3) separation and detection of the immune complex by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. Both polyclonal and monoclonal antibodies can be used to immunoprecipitate the antigen. Polyclonal antibodies usually bind to multiple sites on the antigen and, therefore, are more efficient than monoclonal antibodies. The advantage of using monoclonal antibodies for immunoprecipitations is the specificity of the antibody-antigen interaction because monoclonal antibodies bind to only one epitope of the antigen. If the antigen of interest is tyrosine phosphorylated, and this tyrosine phosphorylation is under investigation, each step has to be handled with extra care to ensure success of the procedure. The buffer used to lyse the cells depends on the nature of the antigen, with more stringent conditions required for integral membrane proteins than for soluble cytosolic proteins. In either case, it is very important to add the protease inhibitors to the lysis buffer in order to prevent degradation of the antigen.

Occludin is an integral membrane protein that is tyrosine phosphorylated when localized at tight junctions. When Ca(2+) was depleted from the culture medium, occludin tyrosine phosphorylation was diminished from Madin-Darby canine kidney epithelial cells in 2 min. This dephosphorylation was correlated with a significant reduction in transepithelial electrical resistance (TER), indicating a global loss of the tight junction barrier function. Reconstitution of Ca(2+) resulted in a robust tyrosine rephosphorylation of occludin that was temporally associated with an increase in TER. Moreover, we demonstrate in this study that occludin was colocalized with the nonreceptor tyrosine kinase c-Yes at cell junction areas and formed an immunoprecipitable complex with c-Yes in vivo. This complex dissociated when the cells were incubated in medium without Ca(2+) or treated with a c-Yes inhibitor, CGP77675. In the presence of CGP77675 after Ca(2+) repletion, occludin tyrosine phosphorylation was completely abolished and both tight junction formation and the increase of the TER were inhibited. Our study thus provides strong evidence that occludin tyrosine phosphorylation is tightly linked to tight junction formation in epithelial cells, and that the nonreceptor tyrosine kinase c-Yes is involved in the regulation of this process.

The tight junction protein claudin‐7 is essential for tight junction function and intestinal homeostasis. Cldn7 deletion in mice leads to an inflammatory bowel disease‐like phenotype exhibiting severe intestinal epithelial damage, weight loss, inflammation, mucosal ulcerations, and epithelial hyperplasia. Claudin‐7 has also been shown to be involved in cancer metastasis and invasion. Here, we test our hypothesis that claudin‐7 plays an important role in regulating colonic intestinal stem cell function. Conditional knockout of Cldn7 in the colon led to impaired epithelial cell differentiation, hyperproliferative epithelium, a decrease in active stem cells, and dramatically altered gene expression profiles. In 3D colonoid culture, claudin‐7–deficient crypts were unable to survive and form spheroids, emphasizing the importance of claudin‐7 in stem cell survival. Inhibition of the Hippo pathway or activation of Notch signaling partially rescued the defective stem cell behavior. Concurrent Notch activation and Hippo inhibition resulted in restored colonoid survival, growth, and differentiation to the level comparable to those of wild‐type derived crypts. In this study, we highlight the essential role of claudin‐7 in regulating Notch and Hippo signaling–dependent colonic stem cell functions, including survival, self‐renewal, and differentiation. These new findings may shed light on potential avenues to explore for drug development in colorectal cancer. [ABSTRACT FROM AUTHOR]

Epithelial cells are polarized with defined apical tight junctions (TJs), lateral adherens junctions (AJs), and basal integrin–matrix interactions. However, it is increasingly recognized that resident cell junction proteins can be found in varying locations and with previously unrecognized functions. Our study here presents the nanoarchitecture and nanocolocalization of cell junction proteins in culture and tissue by stochastic optical reconstruction microscopy (STORM). The Z‐axial view of noncancerous MDCK‐II and PZ‐HPV‐7 cell–cell junctions resolved β‐catenin and p120ctn localizations to TJs and AJs, with p120ctn apical to β‐catenin and colocalizing with TJ protein claudin‐7. More basally, p120ctn and β‐catenin become colocalized. This topography was lost in isogenic Ras‐transformed MDCK cells and cancerous PC3 cells, where p120ctn becomes basally localized in relation to β‐catenin. Claudin‐7 gene conditional knockout (cKO) in mice also have altered polarity of p120ctn relative to β‐catenin, like that seen in normal‐to‐cancer cell phenotypic transformation. Additionally, claudin‐7 cKO resulted in redistribution and relocalization of other cell junction proteins, including claudin‐1, zonula occludens‐1, integrin α2, epithelial cell adhesion molecule, and focal adhesion kinase (FAK); specifically, integrin α2 and FAK were observed at the apical–lateral compartment. Our data show that STORM reveals regional cellular junction nanoarchitecture previously uncharacterized, providing new insight into potential trans‐compartmental modulation of protein functions. [ABSTRACT FROM AUTHOR]

Claudins are a family of tight junction proteins, and serve important roles in epithelial barrier, selective ion transports and cancer metastasis. Although the exact role of claudin-7 in human lung cancer has not been completely elucidated, recent clinical studies have demonstrated that claudin-7 is associated with the survival of patients with lung cancer. Our previous studies have demonstrated that claudin-7 forms a protein complex with integrin β1 in human lung cancer cells. The knockdown (KD) of claudin-7 by short hairpin RNA (shRNA) reduced integrin β1 expression and increased the cell proliferative rate, whereas claudin-7 re-expression in the KD cells decreased the cell proliferation. It is unknown as to whether claudin-7 and integrin β1 regulate cell proliferation and invasion synergistically or independently. In the present study, it was observed that ectopic expression of integrin β1 in claudin-7 KD lung cancer cells did not reduce the cell proliferation. However, integrin β1-transfected cells migrated more effectively in wound healing and cell invasion assays and were more adhesive in a cell attachment assay when compared with those of claudin-7 KD cells. This indicates that claudin-7 controls cell proliferation, while cell attachment and motility were regulated partially through integrin β1. Additionally, claudin-7 overexpression in claudin-7 KD cells resulted in an improved ability to attach to the surface of cell culture plates and a higher expression of focal adhesion proteins when compared with claudin-7 non-KD control cells, which supports the role of claudin-7 in cell adhesion and motility. Taken together, these data suggest that claudin-7 regulates cell motility through integrin β1, providing additional insight into the roles of claudins in carcinogenesis and cancer cell metastasis. [ABSTRACT FROM AUTHOR]

Background: As a potential tumor suppressor gene, Claudin-7 (Cldn7), which is a component of tight junctions, may play an important role in colorectal cancer occurrence and development.Aims: To generate a knockout mouse model of inducible conditional Cldn7 in the intestine and analyze the phenotype of the mice after induction with tamoxifen.Methods: We constructed Cldn7-flox transgenic mice and crossed them with Villin-CreERT2 mice. The Cldn7 inducible conditional knockout mice appeared normal and were well developed at birth. We induced Cldn7 gene deletion by injecting different dosages of tamoxifen into the mice and then conducted a further phenotypic analysis.Results: After induction for 5 days in succession at a dose of 200 µl tamoxifen in sunflower oil at 10 mg/ml per mouse every time, the mice appeared dehydrated, had a lower temperature, and displayed inactivity or death. The results of hematoxylin-eosin staining showed that the intestines of the Cldn7 inducible conditional knockout mice had severe intestinal defects that included epithelial cell sloughing, necrosis, inflammation and hyperplasia. Owing to the death of ICKO mice, we adjusted the dose of tamoxifen to a dose of 100 µl in sunflower oil at 10 mg/ml per mouse (aged more than 8 weeks old) every 4 days. And we could induce atypical hyperplasia and adenoma in the intestine. Immunofluorescent staining indicated that the intestinal epithelial structure was destroyed. Electron microscopy experimental analysis indicated that the intercellular gap along the basolateral membrane of Cldn7 inducible conditional knockout mice in the intestine was increased and that contact between the cells and matrix was loosened.Conclusions: We generated a model of intestinal Cldn7 inducible conditional knockout mice. Intestinal Cldn7 deletion induced by tamoxifen initiated inflammation and hyperplasia in mice. [ABSTRACT FROM AUTHOR]

Bartter's syndrome is a constellation of symptoms characterized by hyper-reninemic hypokalemia, metabolic alkalosis, elevated renin and aldosterone, low or normal blood pressure, and hyperplasia of the juxtaglomerular apparatus. So far, five gene mutations in proteins regulating the sodium chloride transport in the thick ascending limb of Henle's loop have been described. However, the molecular mechanisms underlying the presentation of hypomagnesemia in some of these patients remains unclear. Claudins are a family of transmembranous proteins within the tight junctions that have been shown to be important for the paracellular movement of ions. Mutations in claudin-16 have been identified in patients with familial hypomagnesemia with hypercalciuria and nephrocalcinosis. To test the hypothesis that mutations in claudin genes may be involved in the altered magnesium and calcium transport in Bartter's syndrome, we began to examine the genes of claudins known to be present in renal tubules in four pediatric patients with Bartter's syndrome. All four patients were African Americans with hypomagnesemia and hypercalciuria. In this study, we did not find any mutation in the coding regions of claudin-2, -3, -4, -7, -8, -10, -11, or -16 genes in these patients. However, all patients had a single nucleotide substitution of C for T at the position of 451 of claudin-8 gene sequence that changes amino acid residue from serine to proline at the position of 151 in the second extracellular domain of claudin-8 protein. The significance of this known single nucleotide polymorphism remains to be determined. [ABSTRACT FROM AUTHOR]

Claudin-7 knockout (CLDN7−/−) mice display renal salt wasting and dehydration phenotypes. To address the role of CLDN7 in kidneys, we established collecting duct (CD) cell lines from CLDN7+/+ and CLDN7−/− mouse kidneys. We found that deletion of CLDN7 increased the transepithelial resistance (TER) and decreased the paracellular permeability for Cl− and Na+ in CLDN7−/− CD cells. Inhibition of transcellular Cl− and Na+ channels has no significant effect on TER or dilution potentials. Current-voltage curves were linear in both CLDN7+/+ and CLDN7−/− CD cells, indicating that the ion flux was through the paracellular pathway. The impairment of Cl− and Na+ permeability phenotype can be rescued by CLDN7 re-expression. We also found that WNK4 (its mutations lead to hypertension) expression, but not WNK1, was significantly increased in CLDN7−/− CD cell lines as well as in primary CLDN7−/− CD cells, suggesting that the expression of WNK4 was modulated by CLDN7. In addition, deletion of CLDN7 upregulated the expression level of the apical epithelial sodium channel (ENaC), indicating a potential cross-talk between paracellular and transcellular transport systems. This study demonstrates that CLDN7 plays an important role in salt balance in renal CD cells and modulating WNK4 and ENaC expression levels that are vital in controlling salt-sensitive hypertension. [ABSTRACT FROM AUTHOR]

Abstract: Tight junctions are the most apical component of the junctional complex critical for epithelial cell barrier and polarity functions. Although its disruption is well documented during cancer progression such as epithelial–mesenchymal transition, molecular mechanisms by which tight junction integral membrane protein claudins affect this process remain largely unknown. In this report, we found that claudin-7 was normally expressed in bronchial epithelial cells of human lungs but was either downregulated or disrupted in its distribution pattern in lung cancer. To investigate the function of claudin-7 in lung cancer cells, we transfected claudin-7 cDNA into NCI-H1299, a human lung carcinoma cell line that has no detectable claudin-7 expression. We found that claudin-7 expressing cells showed a reduced response to hepatocyte growth factor (HGF) treatment, were less motile, and formed fewer foot processes than the control cells did. In addition, cells transfected with claudin-7 dramatically decreased their invasive ability after HGF treatment. These effects were mediated through the MAPK signaling pathway since the phosphorylation level of ERK1/2 was significantly lower in claudin-7 transfected cells than in control cells. PD98059, a selective inhibitor of ERK/MAPK pathway, was able to block the motile effect. Claudin-7 formed stable complexes with claudin-1 and -3 and was able to recruit them to the cell-cell junction area in claudin-7 transfected cells. When control and claudin-7 transfected cells were inoculated into nude mice, claudin-7 expressing cells produced smaller tumors than the control cells. Taken together, our study demonstrates that claudin-7 inhibits cell migration and invasion through ERK/MAPK signaling pathway in response to growth factor stimulation in human lung cancer cells. [Copyright &y& Elsevier]

Abstract: Tight junctions (TJ) constitute paracellular diffusion channels regulating the passage of ions and solutes across epithelia. We recently demonstrated that overexpression of the TJ membrane protein claudin-7 in LLC-PK1 cells decreases paracellular permeability to Cl− and increases paracellular permeability to Na+. To investigate the effect of charged amino acid residues in extracellular domains (ED) of claudin-7 on paracellular charge selectivity, we created claudin-7 mutants by replacing negatively charged amino acids on ED with positively charged amino acids. Immunofluorescence light microscopy showed that these mutant proteins were correctly targeted to the cell junction. Ultrastructure examination of TJ morphology did not reveal any difference between cells expressing wildtype (WT) and mutant claudin-7. However, electrophysiological studies showed increased Cl− permeability in cells expressing first extracellular domain (ED1) mutants, but not second extracellular domain (ED2) mutants, compared to that of WT claudin-7. Our results demonstrate that negatively charged amino acids in ED1 of claudin-7 are involved in modulating paracellular Cl− permeability. [Copyright &y& Elsevier]