Desmoglein2 Regulates Claudin2 Expression by Sequestering PI-3-Kinase in Intestinal Epithelial Cells

Inflammation-induced reduction of intestinal desmosomal cadherin Desmoglein 2 (Dsg2) is linked to changes of tight junctions (TJ) leading to impaired intestinal epithelial barrier (IEB) function by undefined mechanisms. We characterized the interplay between loss of Dsg2 and upregulation of pore-forming TJ protein Claudin2. Intraperitoneal application of Dsg2-stablising Tandem peptide (TP) attenuated impaired IEB function, reduction of Dsg2 and increased Claudin2 in DSS-induced colitis in C57Bl/6 mice. TP blocked loss of Dsg2-mediated adhesion and upregulation of Claudin2 in Caco2 cells challenged with TNFα. In Dsg2-deficient Caco2 cells basal expression of Claudin2 was increased which was paralleled by reduced transepithelial electrical resistance and by augmented phosphorylation of AKTSer473 under basal conditions. Inhibition of phosphoinositid-3-kinase proved that PI-3-kinase/AKT-signaling is critical to upregulate Claudin2. In immunostaining PI-3-kinase dissociated from Dsg2 under inflammatory conditions. Immunoprecipitations and proximity ligation assays confirmed a direct interaction of Dsg2 and PI-3-kinase which was abrogated following TNFα application. In summary, Dsg2 regulates Claudin2 expression by sequestering PI-3-kinase to the cell borders in intestinal epithelium.

Effects of 1,2-Dimethylhydrazine on Barrier Properties of Rat Large Intestine and IPEC-J2 Cells

Colon cancer is accompanied by a decrease of epithelial barrier properties, which are determined by tight junction (TJ) proteins between adjacent epithelial cells. The aim of the current study was to analyze the expression of TJ proteins in a rat model of 1,2-dimethylhydrazine (DMH)-induced colorectal cancer, as well as the barrier properties and TJ protein expression of IPEC-J2 cell monolayers after incubation with DMH. Transepithelial electrical resistance and paracellular permeability for sodium fluorescein of IPEC-J2 were examined by an epithelial volt/ohm meter and spectrophotometry. The expression and localization of TJ proteins were analyzed by immunoblotting and immunohistochemistry. In the colonic tumors of rats with DMH-induced carcinogenesis, the expression of claudin-3 and -4 was significantly increased compared to controls. The transepithelial electrical resistance of IPEC-J2 cells increased, while paracellular permeability for sodium fluorescein decreased, accompanied by an increased expression of claudin-4. The increase of claudin-4 in rat colon after chronic DMH exposure was consistent with the acute effect of DMH on IPEC-J2 cells, which may indicate an essential role of this protein in colorectal cancer development.

Protective Effect of QiOne® 2 Pro on Cultured Intestinal Epithelial Cells after Mobile Phone Radiation

QiOne® 2 Pro is a specific device which creates a static field that stimulates water molecules to undergo a transition into the coherent state. Since our body consists of about 70 to 85% of water (depending on age), this coherent state of the water molecules might increase the cellular resistance against exogenous reliabilities such as electromagnetic fields. In this study, the protective effect of QiOne® 2 Pro against mobile phone radiation was examined by using the cultured intestinal epithelial cells. Unprotected cells and untreated control cells served as point of reference. The cell regeneration process as well as the integrity of the intestinal epithelial barrier was investigated by measuring the transepithelial electrical resistance. Mobile phone radiation caused a reduced cell regenerative activity by approximately 60%, whereas the values were about 15% for QiOne® 2 Pro protected cells and untreated controls, respectively. Moreover, mobile phone radiation caused a rupture on the epithelial barrier in unprotected cells by cell death caused due to the oxidative stress with a complete loss of morphological integrity on the barrier. In contrast, untreated controls and QiOne® 2 Pro protected cells did not show any morphological change on the cell layers with an epithelial barrier of a 10-fold higher transepithelial electrical resistance than the unprotected cells. Overall the results clearly demonstrate the sensitivity of intestinal barrier against oxidative stress generated by mobile phone radiation. In addition, the results also show that the QiOne® 2 Pro device is able to reduce unwanted cellular effects of mobile phone radiation.

Beneficial Effects of Quercetin on Microcystin-LR Induced Tight Junction Defects

Quercetin has numerous functions including antioxidant and anti-inflammatory effects. The beneficial effect of quercetin against microcystin-LR (MC-LR)-induced testicular tight junctions (TJs) defects in vitro and in vivo were investigated. Significant reductions in transepithelial electrical resistance, occludin, and zonula occludens-1(ZO-1) levels were detected in the MC-LR-treated TM4 cells, and quercetin attenuated these effects. Interestingly, quercetin suppressed MC-LR-induced phosphorylation of protein kinase B (AKT). It effectively inhibited the accumulation of reactive oxygen species (ROS) in cells stimulated by MC-LR. In addition, ROS inhibitors blocked the TJ damage that is dependent on the AKT signaling pathway induced by MC-LR. In conclusion, our results suggest that alleviates MC-LR-impaired TJs by suppressing the ROS-regulated activation of the AKT pathway.

Measurements of transepithelial electrical resistance (TEER) are affected by junctional length in immature epithelial monolayers

The measurement of transepithelial electrical resistance (TEER) is a common technique to determine the barrier integrity of epithelial cell monolayers. However, it is remarkable that absolute TEER values of similar cell types cultured under comparable conditions show an immense heterogeneity. Based on previous observations, we hypothesized that the heterogeneity of absolute TEER measurements can not only be explained by maturation of junctional proteins but rather by dynamics in the absolute length of cell junctions within monolayers. Therefore, we analyzed TEER in epithelial cell monolayers of Caco2 cells during their differentiation, with special emphasis on both changes in the junctional complex and overall cell morphology within monolayers. We found that in epithelial Caco2 monolayers TEER increased until confluency, then decreased for some time, which was then followed by an additional increase during junctional differentiation. In contrast, permeability of macromolecules measured at different time points as 4 kDA fluorescein isothiocyanate (FITC)-dextran flux across monolayers steadily decreased during this time. Detailed analysis suggested that this observation could be explained by alterations of junctional length along the cell borders within monolayers during differentiation. In conclusion, these observations confirmed that changes in cell numbers and consecutive increase of junctional length have a critical impact on TEER values, especially at stages of early confluency when junctions are immature.

Heterogeneity of the barrier properties of the colon in rat

The incidence of colorectal cancer in different parts of the large intestine is not the same, as tumors more often appear in the distal part of the colon compared to the proximal one. The purpose of this study was to investigate heterogeneity of the barrier properties of the colon and clarify the effects of Prostaglandin E2 and interleukin-1beta on its different parts. An in-depth analysis of short circuit current, transepithelial electrical resistance and paracellular permeability for sodium fluorescein in Ussing chambers showed that the proximal part of the colon was less permeable compared to the distal one and the substances had different effects on the parameters of permeability in different parts of the colon. We suppose that heterogeneity of the barrier properties of the colon and various effects of their regulation by local molecular agents may determine different incidence of pathologies in the colon.

Epithelial Barrier Integrity Profiling: Combined Approach Using Cellular Junctional Complex Imaging and Transepithelial Electrical Resistance

A core aspect of epithelial cell function is barrier integrity. A loss of barrier integrity is a feature of a number of respiratory diseases, including asthma, allergic rhinitis, and chronic obstructive pulmonary disease. Restoration of barrier integrity is a target for respiratory disease drug discovery. Traditional methods for assessing barrier integrity have their limitations. Transepithelial electrical resistance (TEER) and dextran permeability methods can give poor in vitro assay robustness. Traditional junctional complex imaging approaches are labor-intensive and tend to be qualitative but not quantitative. To provide a robust and quantitative assessment of barrier integrity, high-content imaging of junctional complexes was combined with TEER. A scalable immunofluorescent high-content imaging technique, with automated quantification of junctional complex proteins zonula occludens-1 and occludin, was established in 3D pseudostratified primary human bronchial epithelial cells cultured at an air–liquid interface. Ionic permeability was measured using TEER on the same culture wells. The improvements to current technologies include the design of a novel 24-well holder to enable scalable in situ confocal cell imaging without Transwell membrane excision, the development of image analysis pipelines to quantify in-focus junctional complex structures in each plane of a Z stack, and the enhancement of the TEER data analysis process to enable statistical evaluation of treatment effects on barrier integrity. This novel approach was validated by demonstrating measurable changes in barrier integrity in cells grown under conditions known to perturb epithelial cell function.

High throughput transepithelial electrical resistance (TEER) measurements on perfused membrane-free epithelia

We present an instrument for simultaneously measuring TEER in up to 80 perfused epithelial tubules on an OrganoPlate. The sensitivity, speed and ease of use enables screening of tubules during formation, drug exposure and inflammatory processes.