Mfsd2a regulates the blood-labyrinth-barrier formation and function through tight junctions and transcytosis.

The Blood-Labyrinth Barrier (BLB) is pivotal for the maintenance of lymphatic homeostasis within the inner ear, yet the intricacies of its development and function are inadequately understood. The present investigation delves into the contribution of the Mfsd2a molecule, integral to the structural and functional integrity of the Blood-Brain Barrier (BBB), to the ontogeny and sustenance of the BLB. Our empirical findings delineate that the maturation of the BLB in murine models is not realized until approximately two weeks post-birth, with preceding stages characterized by notable permeability. Transcriptomic analysis elucidates a marked augmentation in Mfsd2a expression within the lateral wall of the cochlea in specimens exhibiting an intact BLB. Moreover, both in vitro and in vivo assays substantiate that a diminution in Mfsd2a expression detrimentally impacts BLB permeability and structural integrity, principally via the attenuation of tight junction protein expression and the enhancement of endothelial cell transcytosis. These insights underscore the indispensable role of Mfsd2a in ensuring BLB integrity and propose it as a viable target for therapeutic interventions aimed at the amelioration of hearing loss.

Heterogenous Gene Expression of Bicellular and Tricellular Tight Junction-Sealing Components in the Human Intestinal Tract.

A major site for the absorption of orally administered drugs is the intestinal tract, where the mucosal epithelium functions as a barrier separating the inside body from the outer environment. The intercellular spaces between adjacent epithelial cells are sealed by bicellular and tricellular tight junctions (TJs). Although one strategy for enhancing intestinal drug absorption is to modulate these TJs, comprehensive gene (mRNA) expression analysis of the TJs components has never been fully carried out in humans. In this study, we used human biopsy samples of normal-appearing mucosa showing no endoscopically visible inflammation collected from the duodenum, jejunum, ileum, colon, and rectum to examine the mRNA expression profiles of TJ components, including occludin and tricellulin and members of the claudin family, zonula occludens family, junctional adhesion molecule (JAM) family, and angulin family. Levels of claudin-3, -4, -7, -8, and -23 expression became more elevated in each segment along the intestinal tract from the upper segments to the lower segments, as did levels of angulin-1 and -2 expression. In contrast, expression of claudin-2 and -15 was decreased in the large intestine compared to the small intestine. Levels of occludin, tricellulin, and JAM-B and -C expression were unchanged throughout the intestine. Considering their segment specificity, claudin-8, claudin-15, and angulin-2 appear to be targets for the development of permeation enhancers in the rectum, small intestine, and large intestine, respectively. These data on heterogenous expression profiles of intestinal TJ components will be useful for the development of safe and efficient intestinal permeation enhancers.

Modulation of intestinal epithelial permeability by chronic small intestinal helminth infections.

Increased permeability of the intestinal epithelial layer is linked to the pathogenesis and perpetuation of a wide range of intestinal and extra-intestinal diseases. Infecting humans with controlled doses of helminths, such as human hookworm (termed hookworm therapy), is proposed as a treatment for many of the same diseases. Helminths induce immunoregulatory changes in their host which could decrease epithelial permeability, which is highlighted as a potential mechanism through which helminths treat disease. Despite this, the influence of a chronic helminth infection on epithelial permeability remains unclear. This study uses the chronically infecting intestinal helminth Heligmosomoides polygyrus to reveal alterations in the expression of intestinal tight junction proteins and epithelial permeability during the infection course. In the acute infection phase (1 week postinfection), an increase in intestinal epithelial permeability is observed. Consistent with this finding, jejunal claudin-2 is upregulated and tricellulin is downregulated. By contrast, in the chronic infection phase (6 weeks postinfection), colonic claudin-1 is upregulated and epithelial permeability decreases. Importantly, this study also investigates changes in epithelial permeability in a small human cohort experimentally challenged with the human hookworm, Necator americanus. It demonstrates a trend toward small intestinal permeability increasing in the acute infection phase (8 weeks postinfection), and colonic and whole gut permeability decreasing in the chronic infection phase (24 weeks postinfection), suggesting a conserved epithelial response between humans and mice. In summary, our findings demonstrate dynamic changes in epithelial permeability during a chronic helminth infection and provide another plausible mechanism by which chronic helminth infections could be utilized to treat disease.

Impaired Intestinal Permeability of Tricellular Tight Junctions in Patients with Irritable Bowel Syndrome with Mixed Bowel Habits (IBS-M).

BACKGROUND: The underlying pathophysiology of irritable bowel syndrome (IBS) is still unclear. Our aim was to investigate the pathophysiological mechanisms of diarrhea, constipation, and antigen uptake in mixed-type IBS (IBS-M). METHODS: Colonoscopic biopsies were obtained from IBS-M patients. Epithelial transport and barrier function of colonic mucosae were characterized in Ussing chambers using impedance spectroscopy. Mucosal permeability to macromolecules was measured. Western blotting for tight junction (TJ) proteins was performed and their subcellular localization was visualized by confocal microscopy. RNA-sequencing was performed for gene expression and signaling pathway analysis. RESULTS: In IBS-M, epithelial resistance and ENaC-dependent sodium absorption were unchanged, while short-circuit current reflecting chloride secretion was reduced. Concomitantly, epithelial permeability for fluorescein and FITC-dextran-4000 increased. TJ protein expression of occludin decreased, whereas claudins were unaltered. Confocal microscopy revealed the de-localization of tricellulin from tricellular TJs. Involved pathways were detected as proinflammatory cytokine pathways, LPS, PGE2, NGF, and vitamin D. CONCLUSIONS: Decreased anion secretion explains constipation in IBS-M, while ion permeability and sodium absorption were unaltered. Reduced occludin expression resulted in the delocalization of tricellulin from the tricellular TJ, leading to increased macromolecular permeability that contributes to antigen influx into the mucosa and perpetuates a low-grade inflammatory process.

Effects of TAMP family on the tight junction strand network and barrier function in epithelial cells.

Occludin, tricellulin, and marvelD3 belong to the tight junction (TJ)-associated MARVEL protein family. Occludin and tricellulin jointly contribute to TJ strand branching point formation and epithelial barrier maintenance. However, whether marvelD3 has the same function remains unclear. Furthermore, the roles of the carboxy-terminal cytoplasmic tail, which is conserved in occludin and tricellulin, on the regulation of TJ strand morphology have not yet been explored in epithelial cells. We established tricellulin/occludin/marveld3 triple-gene knockout (tKO) MDCK II cells and evaluated the roles of marvelD3 in the TJ strand structure and barrier function using MDCK II cells and a mathematical model. The complexity of TJ strand networks and paracellular barrier did not change in tKO cells compared to that in tricellulin/occludin double-gene knockout (dKO) cells. Exogenous marvelD3 expression in dKO cells did not increase the complexity of TJ strand networks and epithelial barrier tightness. The expression of the carboxy-terminal truncation mutant of tricellulin restored the barrier function in the dKO cells, whereas occludin lacking the carboxy-terminal cytoplasmic tail was not expressed on the plasma membrane. These data suggest that marvelD3 does not affect the morphology of TJ strands and barrier function in MDCK II cells and that the carboxy-terminal cytoplasmic tail of tricellulin is dispensable for barrier improvement.

Differential Expression of the Genes Coding for Adipokines and Epithelial Cell Polarity Components in Women With Low and High Mammographic Density.

BACKGROUND: Women with extensive mammographic density (MD) are more likely to develop breast cancer than women with low MD because of a high epithelial component associated with a high proportion of stromal cells. To elucidate the biological association between high MD and risk of breast cancer, we compared the expression of a panel of genes coding for leptin, adiponectin, and some component of cell polarity and adherens junction complexes in dense and non-dense breast tissue. METHODS: We interrogated a public dataset composed by 120 specimens of normal breast tissue with MD evaluation. The differential expression of the selected genes in the 2 MD subgroups was assessed by the Wilcoxon test, whereas Kruskal-Wallis test evaluated the differential expression of single genes in the fatty, epithelium, or nonfatty compartment. Spearman's correlation measured the relationship among genes in the subset with the highest epithelium proportion. RESULTS: In high MD, the expression level of PARD6B, CRB3, PATJ, LLGL2, CDH1, and MARVELD2 significantly lowered in tissues with the highest epithelium proportion, whereas, in low MD, the expression level of the genes increased with the increasing of the epithelium proportion. In the low MD subgroup, LEP correlated negatively with PRKCZ and DLG3, whereas, in high MD, such correlation was not observed. CONCLUSIONS: The expression of the genes governing cell polarity establishment and cell-cell adhesion assembly differed significantly in the epithelial component of dense and non-dense breasts. The correlation pattern between LEP and PRKCZ or DLG3 agrees with the role of leptin in cell polarity disruption.

Low-density cell culture enhances hepatic function through tight junction formation in HepG2 cells.

BACKGROUND INFORMATION: An in vitro evaluation system using cultured hepatocytes is the most useful method in preclinical research, such as drug metabolism and toxicity test. Human hepatocytes should be used in an in vitro evaluation system because the expression of drug-metabolizing enzymes varies among animal species. HepG2 cells, a liver cancer-derived cell line, are widely used as a human hepatocyte model; however, their hepatic functions are generally weak. RESULTS: In this study, we showed that low-density HepG2 cell culture induces hepatic function. The morphology of HepG2 cells was altered depending on the cell density at the time of seeding. Low-density cultured HepG2 cells proliferated as tightly packed colonies. The HepG2 cell colonies in low-density culture demonstrated enhanced tight junction formation. Tight junction protein gene expression levels, such as those of zonula occludens-1 (ZO-1), junctional adhesion molecule 1 (JAM), claudin, occludin, and tricellulin, increased in low-density cultured HepG2 cells. Phases I and II metabolic enzymes, phase III transporter gene expression, and CYP3A4 activity also increased in low-density cultured HepG2 cells. Occludin and tricellulin knockdown inhibited the increased hepatic function in low-density cultures. Tricellulin knockdown reduced the expression of hepatocyte nuclear factor 6 (HNF6), CCAAT/enhancer-binding protein alpha (CEBPA), and aryl hydrocarbon receptor (AHR). In addition, the expression of nuclear receptor subfamily 1 group h member 2 (NR1H2) increased in low-density cultures, canceled by occludin and tricellulin knockdown. CONCLUSIONS: Our results suggest that low-density HepG2 cell cultures enhance hepatic function by promoting tight junction formation and demonstrate the importance of cell density in drug evaluation using hepatocyte cell lines.

Tricellulin secures the epithelial barrier at tricellular junctions by interacting with actomyosin.

The epithelial cell sheet functions as a barrier to prevent invasion of pathogens. It is necessary to eliminate intercellular gaps not only at bicellular junctions, but also at tricellular contacts, where three cells meet, to maintain epithelial barrier function. To that end, tight junctions between adjacent cells must associate as closely as possible, particularly at tricellular contacts. Tricellulin is an integral component of tricellular tight junctions (tTJs), but the molecular mechanism of its contribution to the epithelial barrier function remains unclear. In this study, we revealed that tricellulin contributes to barrier formation by regulating actomyosin organization at tricellular junctions. Furthermore, we identified α-catenin, which is thought to function only at adherens junctions, as a novel binding partner of tricellulin. α-catenin bridges tricellulin attachment to the bicellular actin cables that are anchored end-on at tricellular junctions. Thus, tricellulin mobilizes actomyosin contractility to close the lateral gap between the TJ strands of the three proximate cells that converge on tricellular junctions.

Occludin and tricellulin facilitate formation of anastomosing tight-junction strand network to improve barrier function.

Tight junctions (TJs) are composed of a claudin-based anastomosing network of TJ strands at which plasma membranes of adjacent epithelial cells are closely attached to regulate the paracellular permeability. Although the TJ proteins occludin and tricellulin have been known to be incorporated in the TJ strand network, their molecular functions remain unknown. Here, we established tricellulin/occludin-double knockout (dKO) MDCK II cells using a genome editing technique and evaluated the structure and barrier function of these cells. In freeze-fracture replica electron microscopy, the TJ strands of tricellulin/occludin-dKO cells had fewer branches and were less anastomosed compared with the controls. The paracellular permeability of ions and small tracers was increased in the dKO cells. A single KO of tricellulin or occludin had limited effects on the morphology and permeability of TJs. Mathematical simulation using a simplified TJ strand network model predicted that reduced cross-links in TJ strands lead to increased permeability of ions and small macromolecules. Furthermore, overexpression of occludin increased the complexity of TJ strand network and strengthened barrier function. Taken together, our data suggest that tricellulin and occludin mediate the formation and/or stabilization of TJ-strand branching points and contribute to the maintenance of epithelial barrier integrity.

Association of tricellulin expression with poor colorectal cancer prognosis and metastasis.

Tricellulin is a tight­junction transmembrane protein that regulates cell­cell interactions. Altered tricellulin expression could promote tumor cell invasions and metastasis in human cancers. The present study assessed tricellulin expression in colorectal cancer tissues for any association with clinicopathological features of colorectal cancer patients and then investigated the underlying molecular events using quantitative proteomic analysis and in vitro experiments. Tissue samples from 98 colorectal cancer patients and 15 volunteers were collected for immunohistochemistry. Colorectal cell lines were used to overexpress or knockdown tricellulin expression in various assays. The data revealed that upregulated tricellulin expression was associated with lymph node and distant metastases and poor prognosis, while tricellulin overexpression promoted colorectal cancer cell migration and invasion in vitro. In contrast, tricellulin knockdown had positive effects on the tumor cells. Furthermore, TMT­LC­MS/MS and bioinformatics analyses revealed that tricellulin was involved in EMT and reduction of apoptosis through the NF­κB signaling pathway. These findings highlight for the first time the significance of tricellulin in colorectal cancer development and progression. Further study may validate tricellulin as a novel biomarker and target for colorectal cancer.

Diesel exhaust particle exposure reduces expression of the epithelial tight junction protein Tricellulin.

BACKGROUND: While exposure to diesel exhaust particles has been linked to aberrant immune responses in allergic diseases such as asthma, little attention has been paid to their effects on the airway epithelial barrier. In this study, we sought to determine the effect of diesel exhaust exposure on airway epithelial barrier function and composition using in vitro and in vivo model systems. METHODS: 16HBE14o- human bronchial epithelial cells were grown on collagen coated Transwell inserts and exposed to 5 to 50 µg/cm2 SRM 2975 diesel particulate matter (DEP) suspended in cell culture medium or vehicle controls. Changes in barrier function were assessed by measuring transepithelial electrical resistance (TEER) and permeability to 4 kDa FITC Dextran. Neonatal BALB/c mice were exposed to aerosolized DEP (255 ± 89 µg/m3; 2 h per day for 5 days) and changes in the tight junction protein Tricellulin were assessed 2 weeks post exposure. RESULTS: A six-hour incubation of epithelial cells with diesel exhaust particles caused a significant concentration-dependent reduction in epithelial barrier integrity as measured by decreased TEER and increased permeability to 4 kDa FITC-Dextran. This reduction in epithelial barrier integrity corresponded to a significant reduction in expression of the tight junction protein Tricellulin. siRNA mediated knockdown of Tricellulin recapitulated changes in barrier function caused by DEP exposure. Neonatal exposure to aerosolized DEP caused a significant reduction in lung Tricellulin 2 weeks post exposure at both the protein and mRNA level. CONCLUSION: Short term exposure to DEP causes a significant reduction in epithelial barrier integrity through a reduction in the tight junction protein Tricellulin. Neonatal exposure to aerosolized DEP caused a significant and sustained reduction in Tricellulin protein and mRNA in the lung, suggesting that early life exposure to inhaled DEP may cause lasting changes in airway epithelial barrier function.

Leptin Downregulates Angulin-1 in Active Crohn's Disease via STAT3.

Crohn's disease (CD) has an altered intestinal barrier function, yet the underlying mechanisms remain to be disclosed. The tricellular tight junction protein tricellulin is involved in the maintenance of the paracellular macromolecule barrier and features an unchanged expression level in CD but a shifted localization. As angulins are known to regulate the localization of tricellulin, we hypothesized the involvement of angulins in CD. Using human biopsies, we found angulin-1 was downregulated in active CD compared with both controls and CD in remission. In T84 and Caco-2 monolayers, leptin, a cytokine secreted by fat tissue and affected in CD, decreased angulin-1 expression. This effect was completely blocked by STAT3 inhibitors, Stattic and WP1066, but only partially by JAK2 inhibitor AG490. The effect of leptin was also seen at a functional level as we observed in Caco-2 cells an increased permeability for FITC-dextran 4 kDa indicating an impaired barrier against macromolecule uptake. In conclusion, we were able to show that in active CD angulin-1 expression is downregulated, which leads to increased macromolecule permeability and is inducible by leptin via STAT3. This suggests that angulin-1 and leptin secretion are potential targets for intervention in CD to restore the impaired intestinal barrier.

Altered Structural Expression and Enzymatic Activity Parameters in Quiescent Ulcerative Colitis: Are These Potential Normalization Criteria?

Mucosal healing determined by endoscopy is currently the remission standard for ulcerative colitis (UC). However, new criteria for remission are emerging, such as histologic normalization, which appears to correlate better to the risk of relapse. Here, we study mucosal healing on a molecular and functional level in quiescent UC. We obtained endoscopic biopsies from 33 quiescent UC patients and from 17 controls. Histology was assessed using Geboes score. Protein and mRNA levels were evaluated for the tight junction proteins claudin-2, claudin-4, occludin, and tricellulin, as well as Cl-/HCO3- exchanger DRA, and cyclo-oxygenase enzymes (COX-1, COX-2). The mucosal activity of COX-1 and COX-2 enzymes was assessed in modified Ussing chambers, measuring electrogenic ion transport (short-circuit current, SCC). Chronic inflammation was present in most UC patients. The protein level of claudin-4 was reduced, while mRNA-levels of claudin-2 and claudin-4 were upregulated in UC patients. Surprisingly, the mRNA level of COX-1 was downregulated, but was unaltered for COX-2. Basal ion transport was not affected, while COX-2 inhibition induced a two-fold larger decrease in SCC in UC patients. Despite being in clinical and endoscopic remission, quiescent UC patients demonstrated abnormal mucosal barrier properties at the molecular and functional level. Further exploration of mucosal molecular signature for revision of current remission standards should be considered.

Tricellulin Effect on Paracellular Water Transport.

In epithelia, large amounts of water pass by transcellular and paracellular pathways, driven by the osmotic gradient built up by the movement of solutes. The transcellular pathway has been molecularly characterized by the discovery of aquaporin membrane channels. Unlike this, the existence of a paracellular pathway for water through the tight junctions (TJ) was discussed controversially for many years until two molecular components of paracellular water transport, claudin-2 and claudin-15, were identified. A main protein of the tricellular TJ (tTJ), tricellulin, was shown to be downregulated in ulcerative colitis leading to increased permeability to macromolecules. Whether or not tricellulin also regulates water transport is unknown yet. To this end, an epithelial cell line featuring properties of a tight epithelium, Madin-Darby canine kidney cells clone 7 (MDCK C7), was stably transfected with small hairpin RNA (shRNA) targeting tricellulin, a protein of the tTJ essential for the barrier against passage of solutes up to 10 kDa. Water flux was induced by osmotic gradients using mannitol or 4 and 40 kDa-dextran. Water flux in tricellulin knockdown (KD) cells was higher compared to that of vector controls, indicating a direct role of tricellulin in regulating water permeability in a tight epithelial cell line. We conclude that tricellulin increases water permeability at reduced expression.

Pyk2-dependent phosphorylation of LSR enhances localization of LSR and tricellulin at tricellular tight junctions.

Tight junctions (TJs) are cellular junctions within the mammalian epithelial cell sheet that function as a physical barrier to molecular transport within the intercellular space. Dysregulation of TJs leads to various diseases. Tricellular TJs (tTJs), specialized structural variants of TJs, are formed by multiple transmembrane proteins (e.g., lipolysis-stimulated lipoprotein receptor [LSR] and tricellulin) within tricellular contacts in the mammalian epithelial cell sheet. However, the mechanism for recruiting LSR and tricellulin to tTJs is largely unknown. Previous studies have identified that tyrphostin 9, the dual inhibitor of Pyk2 (a nonreceptor tyrosine kinase) and receptor tyrosine kinase platelet-derived growth factor receptor (PDGFR), suppresses LSR and tricellulin recruitment to tTJs in EpH4 (a mouse mammary epithelial cell line) cells. In this study, we investigated the effect of Pyk2 inhibition on LSR and tricellulin localization to tTJs. Pyk2 inactivation by its specific inhibitor or repression by RNAi inhibited the localization of LSR and downstream tricellulin to tTJs without changing their expression level in EpH4 cells. Pyk2-dependent changes in subcellular LSR and tricellulin localization were independent of c-Jun N-terminal kinase (JNK) activation and expression. Additionally, Pyk2-dependent LSR phosphorylation at Tyr-237 was required for LSR and tricellulin localization to tTJs and decreased epithelial barrier function. Our findings indicated a novel mechanism by which Pyk2 regulates tTJ assembly and epithelial barrier function in the mammalian epithelial cell sheet.

Apoptotic Fragmentation of Tricellulin.

Apoptotic extrusion of cells from epithelial cell layers is of central importance for epithelial homeostasis. As a prerequisite cell-cell contacts between apoptotic cells and their neighbors have to be dissociated. Tricellular tight junctions (tTJs) represent specialized structures that seal polarized epithelial cells at sites where three cells meet and are characterized by the specific expression of tricellulin and angulins. Here, we specifically addressed the fate of tricellulin in apoptotic cells. METHODS: Apoptosis was induced by staurosporine or camptothecin in MDCKII and RT-112 cells. The fate of tricellulin was analyzed by Western blotting and immunofluorescence microscopy. Caspase activity was inhibited by Z-VAD-FMK or Z-DEVD-FMK. RESULTS: Induction of apoptosis induces the degradation of tricellulin with time. Aspartate residues 487 and 441 were identified as caspase cleavage-sites in the C-terminal coiled-coil domain of human tricellulin. Fragmentation of tricellulin was inhibited in the presence of caspase inhibitors or when Asp487 or Asp441 were mutated to asparagine. Deletion of the tricellulin C-terminal amino acids prevented binding to lipolysis-stimulated lipoprotein receptor (LSR)/angulin-1 and thus should impair specific localization of tricellulin to tTJs. CONCLUSIONS: Tricellulin is a substrate of caspases and its cleavage in consequence contributes to the dissolution of tTJs during apoptosis.

Cholera toxin perturbs the paracellular barrier in the small intestinal epithelium of rats by affecting claudin-2 and tricellulin.

Cholera toxin is commonly known to induce chloride secretion of the intestine. In recent years, effects on epithelial barrier function have been reported, indicating synergistic co-regulation of transporters and tight junction proteins. Our current study focused on the analysis of cholera toxin effects on transepithelial resistance and on tight junction proteins, the latter known as structural correlates of barrier function. Ligated segments of the rat jejunum were injected with buffered solution containing cholera toxin (1 µg/ml) and incubated for 4 h. Subsequently, selfsame tissue specimens were mounted in Ussing chambers, and cholera toxin (1 µg/ml) was added on the apical side. Transepithelial resistance and permeability of sodium fluorescein (376 Da) were analyzed. Subsequently, tissues were removed, expression and localization of claudins were analyzed, and morphological studies were performed employing transmission electron microscopy and confocal laser scanning microscopy. Cholera toxin induced a marked decrease in transepithelial resistance in the rat jejunal epithelium and an increase in paracellular permeability for sodium fluorescein. Immunoblotting of tight junction proteins revealed an increase in claudin-2 signals, which was verified by confocal laser scanning immunofluorescence microscopy, and a decrease in tricellulin, whereas other tight junction proteins remained unchanged. Transmission electron microscopy showed a reduction in the number of microvilli after incubation with cholera toxin. Moreover, cholera toxin led to a widening of the intercellular space between enterocytes. In accordance with the commonly known prosecretory effect of cholera toxin, our study revealed a complementary effect on small intestinal barrier function and integrity, which might constitute a pathomechanism with high relevance for prevention and therapeutic approaches.

Caprate Modulates Intestinal Barrier Function in Porcine Peyer's Patch Follicle-Associated Epithelium.

BACKGROUND: Many food components influence intestinal epithelial barrier properties and might therefore also affect susceptibility to the development of food allergies. Such allergies are triggered by increased antibody production initiated in Peyer's patches (PP). Usually, the presentation of antigens in the lumen of the gut to the immune cells of the PP is strongly regulated by the follicle-associated epithelium (FAE) that covers the PP. As the food component caprate has been shown to impede barrier properties in villous epithelium, we hypothesized that caprate also affects the barrier function of the PP FAE, thereby possibly contributing a risk factor for the development of food allergies. METHODS: In this study, we have focused on the effects of caprate on the barrier function of PP, employing in vitro and ex vivo experimental setups to investigate functional and molecular barrier properties. Incubation with caprate induced an increase of transepithelial resistance, and a marked increase of permeability for the paracellular marker fluorescein in porcine PP to 180% of control values. These effects are in accordance with changes in the expression levels of the barrier-forming tight junction proteins tricellulin and claudin-5. CONCLUSIONS: This barrier-affecting mechanism could be involved in the initial steps of a food allergy, since it might trigger unregulated contact of the gut lumen with antigens.

Tricellulin Expression and its Deletion Effects in the Endolymphatic Sac.

OBJECTIVES: Tricellulin is a tight junction (TJ)-forming protein that participates in the sealing function of tricellular TJs. Tricellulin-knockout (Tric-/-) mice show progressive hearing loss with degeneration of hair cells in the cochlea without physiological or physical disorders. In the present study, we investigated the tricellulin expression and its deletion effects in the endolymphatic sac (ES) using Tric-/- mice. MATERIALS AND METHODS: The ES epithelia from wild-type (WT) mice were laser-microdissected, and RT-PCR was performed. The ES sections from Tric-/- and WT mice were immunostained with an anti-tricellulin antibody. Hematoxylin and eosin staining was performed for morphological examination. The inner ear of Tric-/- mice was perfused with biotinylation reagents, and the ES sections were observed for tracer permeability assay after applying streptavidin-Alexa Fluor 488 conjugate. RESULTS: The tricellulin expression was confirmed by RT-PCR and by immunohistochemistry in the WT ES. The ES in Tric-/- mice showed normal morphology and revealed no biotin leakage from the lumen. CONCLUSION: The ES in Tric-/- mice showed no changes in morphology or disruption in macromolecular barrier function. The effects of solute leakages in the ES of Tric-/- mice may be very limited and compensatable, or that the ES epithelia may have other sealing system covering the lack of tricellulin.