Confluent populations of the epithelial cell line, MDCK II, develop circumferential tight junctions joining adjacent cells to create a barrier to the paracellular movement of solutes and water. Treatment of MDCK II cell populations from the apical surface with 1 mM Na-caprate increased permeability to macromolecules (Leak Pathway) without increasing monolayer disruption or cell death. Graphical analysis of the apparent permeability versus solute Stokes radius for a size range of fluorescein-dextran species indicates apical 1 mM Na-caprate enhances Leak Pathway permeability by increasing the number of Leak Pathway openings without significantly affecting opening size. Na-caprate treatment did not alter the content of any tight junction protein examined. Treatment of MDCK II cell populations with apical 1 mM Na-caprate disrupted basal F-actin stress fibers and decreased the tortuosity of the tight junctions. Treatment of MDCK II cell populations with blebbistatin, a myosin ATPase inhibitor, alone had little effect on Leak Pathway permeability but synergistically increased Leak Pathway permeability when added with 1 mM Na-caprate. Na-caprate exhibited a similar ability to increase Leak Pathway permeability in wild type MDCK II cell monolayers and ZO-1 knockdown MDCK II cell monolayers but an enhanced ability to increase Leak Pathway permeability in monolayers of TOCA-1 knockout MDCK II cells. These results demonstrate that Na-caprate increases MDCK II cell population Leak Pathway permeability by increasing the number of Leak Pathway openings. This action is likely mediated by alterations in F-actin organization, primarily involving disruption of basal F-actin stress fibers.

A general platform for the safe and effective oral delivery of biologics would revolutionize the administration of protein-based drugs, improving access for patients and lowering the financial burden on the health-care industry. Because of their dimensions and physiochemical properties, nanomaterials stand as promising vehicles for navigating the complex and challenging environment in the gastrointestinal (GI) tract. Recent developments have led to materials that protect protein drugs from degradation and enable controlled release in the small intestine, the site of absorption for most proteins. Yet, once present in the small intestine, the protein must transit through the secreted mucus and epithelial cells of the intestinal mucosa into systemic circulation, a process that remains a bottleneck for nanomaterial-based delivery. One attractive pathway through the intestinal mucosa is the paracellular route, which avoids cell trafficking and other degradative processes in the interior of cells. Direct flux between cells is regulated by epithelial tight junctions (TJs) that seal the paracellular space and prevent protein flux. Here, we describe a smart nanoparticle system that directly and transiently disrupts TJs for improved protein delivery, an unrealized goal to-date. We take inspiration from enteropathogenic bacteria that adhere to intestinal epithelia and secrete inhibitors that block TJ interactions in the local environment. To mimic these natural mechanisms, we engineer nanoparticles (EnteroPatho NPs) that attach to the epithelial glycocalyx and release TJ modulators in response to the intestinal pH. We show that EnteroPatho NPs lead to TJ disruption and paracellular protein delivery, giving rise to a general platform for oral delivery.

Tight junctions (TJs) are an important component of cellular connectivity. Claudin family proteins, as a constituent of TJs, determine their barrier properties, cell polarity and paracellular permeability. Claudin-12 is an atypical member of the claudin family, as it belongs to the group of non-classical claudins that lack a PDZ-binding domain. It has been shown that claudin-12 is involved in paracellular Ca2+ transients and it is present in normal and hyperplastic tissues in addition to neoplastic tissues. Dysregulation of claudin-12 expression has been reported in various cancers, suggesting that this protein may play an important role in cancer cell migration, invasion, and metastasis. Some studies have shown that claudin-12 gene functions as a tumor suppressor, but others have reported that overexpression of claudin-12 significantly increases the metastatic properties of various tumor cells. Investigating this dual role of claudin-12 is of utmost importance and should therefore be studied in detail. The aim of this review is to provide an overview of the information available to date on claudin-12, including its structure, expression in various tissues and substances that may affect it, with a final focus on its role in cancer.

Tight junction disruption can lead to pathogenesis of various diseases without therapeutic strategy to recover intestinal barrier integrity. The main objective of this study is to demonstrate the effect of Solanum melongena L. extract (SMLE) on intestinal tight junction recovery and its underlying mechanism. Intestinal barrier function is attenuated by Ca2+ depletion. SMLE treatment increased TER value across T84 cell monolayers. Permeability assay reveals that Ca2+ depletion promotes 4-kDa FITC-dextran permeability, but not 70-kDa FITC-dextran. SMLE suppresses the rate of 4-kDa FITC-dextran permeability, indicating that SMLE inhibits paracellular leak pathway permeability. SMLE-mediated TER increase and leak pathway suppression are abolished by neither calcium/calmodulin-dependent protein kinase kinase β (CaMKKβ) inhibitor nor AMP-activated protein kinase (AMPK) inhibitor. Furthermore, mammalian target of rapamycin (mTOR) and extracellular signal-regulated kinase (ERK) inhibitors have no effects on SMLE-mediated TER increase and leak pathway suppression. Interestingly, SMLE is unable to enhance TER value and diminish leak pathway permeability in T84 cell monolayers pre-treated with sirtuin-1 (SIRT-1) inhibitor. Immunofluorescence staining reveals that SMLE enhances re-assembly of tight junction proteins, including occludin and ZO-1 to intercellular space but this effect is abolished by SIRT-1 inhibitor. These data suggest that SMLE promotes intestinal tight junction re-assembly via SIRT-1-dependent manner.

This work seeks to generate new knowledge about the mechanisms underlying the protective effects of cranberry against urinary tract infections (UTI). Using Caco-2 cells grown in Transwell inserts as an intestinal barrier model, we found that a cranberry-derived digestive fluid (containing 135 ± 5 mg of phenolic compounds/L) increased transepithelial electrical resistance with respect to control (ΔTEER = 54.5 Ω cm2) and decreased FITC-dextran paracellular transport by about 30%, which was related to the upregulation of the gene expression of tight junction (TJ) proteins (i.e., occludin, zonula occludens-1 [ZO-1], and claudin-2) (∼3-4-fold change with respect to control for claudin-2 and ∼2-3-fold for occludin and ZO-1). Similar protective effects, albeit to a lesser extent, were observed when Caco-2 cells were previously infected with uropathogenic Escherichia coli (UPEC). In a urinary barrier model comprising T24 cells grown in Transwell inserts and either noninfected or UPEC-infected, treatments with the cranberry-derived phenolic metabolites 3,4-dihydroxyphenylacetic acid (DOPAC) and phenylacetic acid (PAA) (250 μM) also promoted favorable changes in barrier integrity and permeability. In this line, incubation of noninfected T24 cells with these metabolites induced positive regulatory effects on claudin-2 and ZO-1 expression (∼3.5- and ∼2-fold change with respect to control for DOPAC and ∼1.5- and >2-fold change with respect to control for PAA, respectively). Overall, these results suggest that the protective action of cranberry polyphenols against UTI might involve molecular mechanisms related to the integrity and functionality of the urothelium and intestinal epithelium.

Claudin polymers constitute the tight junction (TJ) backbone that forms paracellular barriers, at least for bigger solutes. While some claudins also seal the barrier for small electrolytes, others form ion channels. For cation-selective claudin-15 and claudin-10b, structural models of channels embedded in homo-polymeric strands have been suggested. Here, we generated a model for the prototypic anion-selective claudin-10a channel. Based on previously established claudin-10b models, dodecamer homology models of claudin-10a embedded in two membranes were analyzed by molecular dynamics simulations. The results indicate that both claudin-10 isoforms share the same strand and channel architecture: Sidewise unsealed tetrameric pore scaffolds are interlocked with adjacent pores via the β1β2 loop of extracellular segment 1. This leads to TJ-like strands with claudin subunits arranged in four joined rows in two opposing membranes. Several but not all cis- and trans-interaction modes are indicated to be conserved among claudin-10a, -10b, and -15. However, pore-lining residues that differ between claudin-10a and -10b (i.e., R33/I35, A34/D36, K69/A71, N54/D56, H60/N62, R62/K64) result in opposite charge selectivity of channels. This was supported by electric field simulations for both claudins and is consistent with previous electrophysiological studies. In summary, for the first time, a structural and mechanistic model of complete and prototypic paracellular anion channels is provided. This improves understanding of epithelial paracellular transport.

Glycosylated mucin proteins contribute to the essential barrier function of the intestinal epithelium. The transmembrane mucin MUC13 is an abundant intestinal glycoprotein with important functions for mucosal maintenance that are not yet completely understood. We demonstrate that in human intestinal epithelial monolayers, MUC13 localized to both the apical surface and the tight junction (TJ) region on the lateral membrane. MUC13 deletion resulted in increased transepithelial resistance (TEER) and reduced translocation of small solutes. TEER buildup in ΔMUC13 cells could be prevented by addition of MLCK, ROCK or protein kinase C (PKC) inhibitors. The levels of TJ proteins including claudins and occludin were highly increased in membrane fractions of MUC13 knockout cells. Removal of the MUC13 cytoplasmic tail (CT) also altered TJ composition but did not affect TEER. The increased buildup of TJ complexes in ΔMUC13 and MUC13-ΔCT cells was dependent on PKC. The responsible PKC member might be PKCδ (or PRKCD) based on elevated protein levels in the absence of full-length MUC13. Our results demonstrate for the first time that a mucin protein can negatively regulate TJ function and stimulate intestinal barrier permeability.

Peritoneal dialysis (PD) and prolonged exposure to PD fluids (PDF) induce peritoneal membrane (PM) fibrosis and hypervascularity, leading to functional PM degeneration. 2-deoxy-glucose (2-DG) has shown potential as PM antifibrotic by inhibiting hyper-glycolysis induced mesothelial-to-mesenchymal transition (MMT). We investigated whether administration of 2-DG with several PDF affects the permeability of mesothelial and endothelial barrier of the PM. The antifibrotic effect of 2-DG was confirmed by the gel contraction assay with embedded mesothelial (MeT-5A) or endothelial (EA.hy926) cells cultured in Dianeal® 2.5 % (CPDF), BicaVera® 2.3 % (BPDF), Balance® 2.3 % (LPDF) with/without 2-DG addition (0.2 mM), and qPCR for αSMA, CDH2 genes. Moreover, 2-DG effect was tested on the permeability of monolayers of mesothelial and endothelial cells by monitoring the transmembrane resistance (RTM), FITC-dextran (10, 70 kDa) diffusion and mRNA expression levels of CLDN-1 to -5, ZO1, SGLT1, and SGLT2 genes. Contractility of MeT-5A cells in CPDF/2-DG was decreased, accompanied by αSMA (0.17 ± 0.03) and CDH2 (2.92 ± 0.29) gene expression fold changes. Changes in αSMA, CDH2 were found in EA.hy926 cells, though αSMA also decreased under LPDF/2-DG incubation (0.42 ± 0.02). Overall, 2-DG mitigated the PDF-induced alterations in mesothelial and endothelial barrier function as shown by RTM, dextran transport and expression levels of the CLDN-1 to -5, ZO1, and SGLT2. Thus, supplementation of PDF with 2-DG not only reduces MMT but also improves functional permeability characteristics of the PM mesothelial and endothelial barrier.

Tight junction proteins play a crucial role in paracellular transport in salivary gland epithelia. It is clear that severe xerostomia in patients with HELIX syndrome is caused by mutations in the claudin-10 gene. However, little is known about the expression pattern and role of claudin-10 in saliva secretion in physical and disease conditions. In the present study, we found that only claudin-10b transcript was expressed in human and mouse submandibular gland (SMG) tissues, and claudin-10 protein was dominantly distributed at the apicolateral membranes of acini in human, rat, and mouse SMGs. Overexpression of claudin-10 significantly reduced transepithelial electrical resistance and increased paracellular transport of dextran and Na+ in SMG-C6 cells. In C57BL/6 mice, pilocarpine stimulation promoted secretion and cation concentration in saliva in a dose-dependent increase. Assembly of claudin-10 to the most apicolateral portions in acini of SMGs was observed in the lower pilocarpine (1 mg/kg)-treated group, and this phenomenon was much obvious in the higher pilocarpine (10 mg/kg)-treated group. Furthermore, 7-, 14-, and 21-wk-old nonobese diabetic (NOD) and BALB/c mice were used to mimic the progression of hyposalivation in Sjögren syndrome. Intensity of claudin-10 protein was obviously lower in SMGs of 14- and 21-wk-old NOD mice compared with that of age-matched BALB/c mice. In the cultured mouse SMG tissues, interferon-γ (IFN-γ) downregulated claudin-10 expression. In claudin-10-overexpressed SMG-C6 cells, paracellular permeability was decreased. Furthermore, IFN-γ stimulation increased p-STAT1 level, whereas pretreatment with JAK/STAT1 antagonist significantly alleviated the IFN-γ-induced claudin-10 downregulation. These results indicate that claudin-10 functions as a pore-forming component in acinar epithelia of SMGs, assembly of claudin-10 is required for saliva secretion, and downregulation of claudin-10 induces hyposecretion. These findings may provide new clues to novel therapeutic targets on hyposalivation.

The gut epithelium is a polarized monolayer that exhibits apical and basolateral membrane surfaces. Monolayer cell components are joined side by side via protein complexes known as tight junction proteins (TJPs), expressed at the most apical extreme of the basolateral membrane. The gut epithelium is a physical barrier that determinates intestinal permeability, referred to as the measurement of the transit of molecules from the intestinal lumen to the bloodstream or, conversely, from the blood to the gut lumen. TJPs play a role in the control of intestinal permeability that can be disrupted by stress through signal pathways triggered by the ligation of receptors with stress hormones like glucocorticoids. Preclinical studies conducted under in vitro and/or in vivo conditions have addressed underlying mechanisms that account for the impact of stress on gut permeability. These mechanisms may provide insights for novel therapeutic interventions in diseases in which stress is a risk factor, like irritable bowel syndrome. The focus of this study was to review, in an integrative context, the neuroendocrine effects of stress, with special emphasis on TJPs along with intestinal permeability.