Tight Junction Assembly Research Articles

When Separation Strengthens Ties

Phase separation underlies functional compartmentalization in living systems. Two recent studies (Beutel et al. and Schwayer et al.) show that zonula occludens (ZO) proteins of tight junctions (TJs) condense into compartments within the cytoplasm that display liquid properties. This ability to condense predicts normal TJ assembly and epithelial barrier function which are essential for vertebrate embryogenesis.

LIMK1/2 are required for actin filament and cell junction assembly in porcine embryos developing in vitro.

ObjectiveThis study was conducted to investigate the roles of LIM kinases (LIMK1 and LIMK2) during porcine early embryo development. We checked the mRNA expression patterns and localization of LIMK1/2 to evaluate their characterization. We further explored the function of LIMK1/2 in developmental competence and their relationship between actin assembly and cell junction integrity, specifically during the first cleavage and compaction.MethodsPig ovaries were transferred from a local slaughterhouse within 1 h and cumulus oocyte complexes (COCs) were collected. COCs were matured in in vitro maturation medium in a CO2 incubator. Metaphase II oocytes were activated using an Electro Cell Manipulator 2001 and microinjected to insert LIMK1/2 dsRNA into the cytoplasm. To confirm the roles of LIMK1/2 during compaction and subsequent blastocyst formation, we employed a LIMK inhibitor (LIMKi3).ResultsLIMK1/2 was localized in cytoplasm in embryos and co-localized with actin in cell-to-cell boundaries after the morula stage. LIMK1/2 knockdown using LIMK1/2 dsRNA significantly decreased the cleavage rate, compared to the control group. Protein levels of E-cadherin and β-catenin, present in adherens junctions, were reduced at the cell-to-cell boundaries in the LIMK1/2 knockdown embryos. Embryos treated with LIMKi3 at the morula stage failed to undergo compaction and could not develop into blastocysts. Actin intensity at the cortical region was considerably reduced in LIMKi3-treated embryos. LIMKi3-induced decrease in cortical actin levels was attributed to the disruption of adherens junction and tight junction assembly. Phosphorylation of cofilin was also reduced in LIMKi3-treated embryos.ConclusionThe above results suggest that LIMK1/2 is crucial for cleavage and compaction through regulation of actin organization and cell junction assembly.

A proteomic study on the protective effect of kaempferol pretreatment against deoxynivalenol-induced intestinal barrier dysfunction in a Caco-2 cell model.

Deoxynivalenol (DON) is one of the most widely distributed mycotoxins in the food chain, and the protective effect of kaempferol (KAM) pretreatment against the barrier dysfunction induced by DON in a Caco-2 cell model was investigated using a proteomic approach. The results showed that after KAM pretreatment, both the numbers of down- and up-regulated differentially expressed proteins were significantly lower than those in the DON group. Gene ontology analysis revealed that differentially expressed proteins were enriched in cell adhesion molecule binding, cell junction, and cell junction assembly. Further study demonstrated that KAM pretreatment affected the expression and assembly of tight junction and adherens junction proteins through the PKA pathway and MAPK/ERK pathway to improve the barrier integrity of the Caco-2 cell monolayer, and nutraceutical approach based on bioactive phytochemicals to improve the body's immunity might be an effective strategy to combat the adverse effects of mycotoxins on intestinal barrier function.

Tight Junction Assembly Ensures Maintenance of Pregnancy during Embryogenesis in a Mouse Model

Recent studies showed that tight junctions (TJs) integrity and assembly are required for blastocyst development in mouse and pig models. However, the biological functions of TJs associated with embryo implantation and maintenance of pregnancy were not investigated yet. To examine whether disrupted TJs affect further embryo development, we employed RNAi approach and inhibitor treatment. The embryos were injected with Cxadr (Coxsackievirus and adenovirus receptor) siRNA for knock down (KD) and treated with Adam10 (A Disintegrin and Metalloproteinase specific inhibitor 10; GI254023X; SI). We compared blastocyst development and paracellular sealing assay using FITC dextran uptake between control and KD or SI embryos. Finally, we transferred control and Cxadr KD or Adam 10 SI treated blastocyst to uteri of recipients. Cxadr KD and Adam 10 SI showed lower blastocyst development and more permeable to FITC-dextran. Moreover, we observed that half of KD and inhibited embryos failed to maintain pregnancies after the second trimester. Our findings suggested that TJs integrity is required for the maintenance of pregnancy and can be used as a selective marker for the successful application of assisted reproduction technologies.

Vasoactive Intestinal Peptide Derived From Liver Mesenchymal Cells Mediates Tight Junction Assembly in Mouse Intrahepatic Bile Ducts.

Formation of intrahepatic bile ducts (IHBDs) proceeds in accordance with their microenvironment. Particularly, mesenchymal cells around portal veins regulate the differentiation and ductular morphogenesis of cholangiocytes in the developing liver; however, further studies are needed to fully understand the arrangement of IHBDs into a continuous hierarchical network. This study aims to clarify the interaction between biliary and liver mesenchymal cells during IHBD formation. To identify candidate factors contributing to this cell–cell interaction, mesenchymal cells were isolated from embryonic day 16.5 matrix metalloproteinase 14 (MMP14)‐deficient (knockout [KO]) mice livers, in which IHBD formation is retarded, and compared with those of the wild type (WT). WT mesenchymal cells significantly facilitated the formation of luminal structures comprised of hepatoblast‐derived cholangiocytes (cholangiocytic cysts), whereas MMP14‐KO mesenchymal cells failed to promote cyst formation. Comprehensive analysis revealed that expression of vasoactive intestinal peptide (VIP) was significantly suppressed in MMP14‐KO mesenchymal cells. VIP and VIP receptor 1 (VIPR1) were mainly expressed in periportal mesenchymal cells and cholangiocytic progenitors during IHBD development, respectively, in vivo. VIP/VIPR1 signaling significantly encouraged cholangiocytic cyst formation and up‐regulated tight junction protein 1, cystic fibrosis transmembrane conductance regulator, and aquaporin 1, in vitro. VIP antagonist significantly suppressed the tight junction assembly and the up‐regulation of ion/water transporters during IHBD development in vivo. In a cholestatic injury model of adult mice, exogenous VIP administration promoted the restoration of damaged tight junctions in bile ducts and improved hyperbilirubinemia. Conclusion: VIP is produced by periportal mesenchymal cells during the perinatal stage. It supports bile duct development by establishing tight junctions and up‐regulating ion/water transporters in cholangiocytes. VIP contributes to prompt recovery from cholestatic damage through the establishment of tight junctions in the bile ducts.

Maternal Yes-Associated Protein Participates in Porcine Blastocyst Development via Modulation of Trophectoderm Epithelium Barrier Function.

The establishment of a functional trophectoderm (TE) epithelium is an essential prerequisite for blastocyst formation and placentation. Transcription coactivator yes-associated protein (YAP), a downstream effector of the hippo signaling pathway, is required for specification of both the TE and epiblast lineages in mice. However, the biological role of YAP in porcine blastocyst development is not known. Here, we report that maternally derived YAP protein is localized to both the cytoplasm and nuclei prior to the morula stage and is then predominantly localized to the TE nuclei in blastocysts. Functionally, maternal YAP knockdown severely impeded blastocyst formation and perturbed the allocation of the first two lineages. The treatment of embryos with verteporfin, a pharmacological inhibitor of YAP, faithfully recapitulated the phenotype observed in YAP deleted embryos. Mechanistically, we found that maternal YAP regulates multiple genes which are important for lineage commitment, tight junction assembly, and fluid accumulation. Consistent with the effects on tight junction gene expression, a permeability assay revealed that paracellular sealing was defective in the trophectoderm epithelium. Lastly, YAP knockdown in a single blastomere at the 2-cell stage revealed that the cellular progeny of the YAP+ blastomere were sufficient to sustain blastocyst formation via direct complementation of the defective trophectoderm epithelium. In summary, these findings demonstrate that maternal YAP facilitates porcine blastocyst development through transcriptional regulation of key genes that are essential for lineage commitment, tight junction assembly, and fluid accumulation.

AMPK Activation Promotes Tight Junction Assembly in Intestinal Epithelial Caco-2 Cells.

The AMP-activated protein kinase (AMPK) is principally known as a major regulator of cellular energy status, but it has been recently shown to play a key structural role in cell-cell junctions. The aim of this study was to evaluate the impact of AMPK activation on the reassembly of tight junctions in intestinal epithelial Caco-2 cells. We generated Caco-2 cells invalidated for AMPK α1/α2 (AMPK dKO) by CRISPR/Cas9 technology and evaluated the effect of the direct AMPK activator 991 on the reassembly of tight junctions following a calcium switch assay. We analyzed the integrity of the epithelial barrier by measuring the trans-epithelial electrical resistance (TEER), the paracellular permeability, and quantification of zonula occludens 1 (ZO-1) deposit at plasma membrane by immunofluorescence. Here, we demonstrated that AMPK deletion induced a delay in tight junction reassembly and relocalization at the plasma membrane during calcium switch, leading to impairments in the establishment of TEER and paracellular permeability. We also showed that 991-induced AMPK activation accelerated the reassembly and reorganization of tight junctions, improved the development of TEER and paracellular permeability after calcium switch. Thus, our results show that AMPK activation ensures a better recovery of epithelial barrier function following injury.

Phase Separation of Zonula Occludens Proteins Drives Formation of Tight Junctions

Tight junctions are cell-adhesion complexes that seal tissues and are involved in cell polarity and signaling. Supra-molecular assembly and positioning of tight junctions as continuous networks of adhesion strands are dependent on the membrane-associated scaffolding proteins ZO1 and ZO2. To understand how zona occludens (ZO) proteins organize junction assembly, we performed quantitative cell biology and invitro reconstitution experiments. We discovered that ZO proteins self-organize membrane-attached compartments via phase separation. Weidentified the multivalent interactions of the conserved PDZ-SH3-GuK supra-domain as the driver of phase separation. These interactions are regulated by phosphorylation and intra-molecular binding. Formation of condensed ZO protein compartments is sufficient to specifically enrich and localize tight-junction proteins, including adhesion receptors, cytoskeletal adapters, and transcription factors. Our results suggest that an active-phase transition of ZO proteins into a condensed membrane-bound compartment drives claudin polymerization and coalescence of a continuous tight-junction belt.

The Cxadr-Adam10 complex plays pivotal roles in tight junction integrity and early trophoblast development in mice.

Understanding preimplantation embryo development has important implications for assisted reproductive technologies (ARTs) after the introduction of in vitro fertilisation and embryo transfer because most embryonic losses occur during pre/peri-implantation. Recent studies have shown that tight junctions (TJs) are important components for embryos to develop to the blastocyst stage. However, their biological function after cavitation has not been extensively studied. We examined TJ assembly focusing on coxsackievirus and adenovirus receptor (Cxadr) and A disintegrin and metalloproteinase 10 (Adam10) using siRNA and/or an Adam10-specific inhibitor (GI254023X). TJ-associated genes, including occludin and tight junction protein 1 (Tjp1), were downregulated in the Cxadr knockdown (KD) embryos but were unaltered in Adam10 KD embryos. However, Adam10 KD or chemical inhibition affected subcellular localisation of Adam10, Cxadr, and Tjp1, leading to disrupted TJ assembly. Furthermore, Cxadr KD or GI254023X-treated blastocysts showed a relatively smaller outgrowth area and aberrant expression of transcription factor AP-2γ, a trophoblast-specific marker in the in vitro embryo outgrowth assay. In summary, we demonstrated that the Cxadr-Adam10 complex might moderate TJ integrity/stability and play pivotal roles during early embryonic development. Collectively, understanding the establishment of the TJ complex and its integrity will provide insight into translational research for predicting and selecting developmental competency for ART.

Gut epithelial and vascular barrier abnormalities in patients with chronic intestinal pseudo-obstruction.

Chronic intestinal pseudo-obstruction (CIPO) is a rare condition due to severe impairment of gut motility responsible for recurrent subocclusive episodes. Although neuromuscular-glial-ICC abnormalities represent the main pathogenetic mechanism, the pathophysiology of CIPO remains poorly understood. Intestinal epithelial and vascular endothelial barrier (IEVB) abnormalities can contribute to neuroepithelial changes by allowing passage of harmful substances. To test retrospectively whether IEVB defects occur in patients with CIPO, we measured the jejunal protein expression of the major tight junction (TJ) components. CIPO patients were subdivided according to gut neuromuscular histopathology: apparently normal (AN); with inflammation (INF); or with degenerative alterations (DEG). The presence of occludin/claudin oligomers (index of TJ assembly), the amount of occludin, claudin-4, and zonula occludens-1 (ZO-1), and the expression of vasoactive intestinal polypeptide (VIP) and glial fibrillary acidic protein (GFAP) immunoreactivities were evaluated on jejunal full-thickness biopsies using Western blot. Oligomers were absent in the 73% of CIPO. Total occludin decreased in CIPO with AN and INF changes. Claudin-4 was upregulated in CIPO with INF and DEG features. ZO-1 and VIP expression decreased selectively in DEG group. GFAP increased in CIPO regardless the histopathological phenotype. The absence of oligomers demonstrated in our study suggests that IEBV is altered in CIPO. The mechanism leading to oligomerization is occludin-dependent in AN and INF, whereas is ZO-1-dependent in DEG. Our study provides support to IEVB abnormalities contributing to CIPO clinical and histopathological features.

Pharmacological stimulation of G–protein coupled receptor 40 alleviates cytokine-induced epithelial barrier disruption in airway epithelial Calu-3 cells

Impairment of airway tight junctions induced by elevated levels of proinflammatory cytokines is implicated in the pathogenesis of inflammatory airway diseases. Pharmacological stimulation of G–protein coupled receptor (GPR) 40, a receptor of polyunsaturated fatty acids, have recently been shown to promote tight junction assembly in airway epithelial cells under non-inflammatory conditions. However, roles of GPR40 in regulating airway epithelial integrity in response to inflammatory insults are unknown. This study was aimed to investigate the effect of GPR40 stimulation on proinflammatory cytokine (TNFα and IL–1β)-induced tight junction disruption in human airway epithelial Calu–3 cells using GW9508, a GPR40 agonist. We found that stimulation of GPR40 by GW9508 attenuated the cytokine–induced airway epithelial barrier leakage as analyzed by measurements of transepithelial electrical resistance and transepithelial flux of fluorescently labeled dextran (molecular weight of 4 kDa). Furthermore, GW9508 prevented the cytokine-induced dislocalization of zonula occludens (ZO)–1, occludin and claudin–1. The barrier-protective effect of GW9508 was abolished by a GPR40 antagonist, but not a GPR120 antagonist. Immunofluorescence staining of NF–ĸB indicated that GW9508 had no effect on cytokine-induced NF–ĸB activation. Intriguingly, GW9508 inhibited cytokine-induced airway epithelial barrier disruption through suppression of extracellular signal–regulated kinase (ERK) phosphorylation in a phospholipase C (PLC) and calcium/calmodulin–dependent protein kinase kinase beta (CaMKKβ)-dependent manner. Collectively, this study uncovered the novel role of GPR40 in preventing cytokine–induced tight junction disruption in airway epithelial cells through mechanisms involving PLC–CaMKKβ-mediated suppression of ERK signaling. Pharmacological stimulation of GPR40 may be beneficial in the treatment of airway diseases.

MON-LB022 Gelesis Novel, Non-Systemic, Superabsorbent Hydrogel Improves Intestinal Barrier Function in Mice with DSS Induced Colitis

BACKGROUND: Intestinal barrier dysfunction exists when the epithelial tight-junctions are disrupted and permit translocation of bacterial lipopolysaccharides into the blood. The resulting endotoxemia cause systemic low-grade inflammation that contributes to the development of comorbidities associated with obesity like type 2 diabetes and NASH. Superabsorbent hydrogels based on the Gelesis platform technology are orally administered and synthesized from crosslinked modified cellulose. Gelesis100, the first in this class, demonstrated significant weight loss in patients with obesity and improvement in insulin sensitivity. We hypothesized that Gel-B with specific mechanical properties, designed to improve intestinal barrier function, would prevent translocation of small molecule into the blood after intestinal barrier injury. METHODS: Groups of 8 to 15 C57BI/6 male mice were administered 3% dextran sodium sulfate (DSS) in drinking water for a total of 6 days, and randomized to either Vehicle, Gel-B (0.5 to 4%) in their food from days 5 to 19. An active control group received anti-p40 IP q3 days on days 6-18. On day 19, all animals were fasted for 4 hours prior to FITC-Dextran dosing. Three hours post dosing, the mice were sacrificed, colon specimen fixed, and blood collected. The incidence of epithelial barrier breaks was assessed with E-cadherin immunohistochemistry. To verify the involvement of epithelial tight junctions, the expression of ZO-1 was assessed in C57BL/6J mice (5 per group) fed chow, for 4 weeks, with or without Gel-B. RESULTS: The level of FITC-Dextran in the serum of mice receiving DSS and vehicle (mean ± SEM, 7.1 ± 4.1) was 3.4 times higher than the naïve mice (2.1 ± 1.1) demonstrating a significant defect in the gut barrier function. In turn, the mice assigned to Gel-B 1% to 4% had meaningful reduction in FITC-Dextran serum levels compared to vehicle treated mice. The highest dose (4%) of Gel-B prevented spilling of dextran into the circulation with serum levels numerically lower than the naïve group with an intact gut barrier (1.4 ± 0.4 compared to 2.1 ± 1.1, respectively). Accordingly, a reduction in epithelial barrier breaks in distal colon samples was observed in the highest dose of Gel-B (0.58 ± 0.15) compared to vehicle treated animals (0.86 ± 0.1). Healthy mice treated with Gel-B (8%) showed increased expression of ZO-1, essential to tight junction assembly between epithelial cells. CONCLUSION: The present study demonstrated that treatment with Gel-B prevented the translocation of small molecule into the circulation indicating an improved intestinal barrier function. This effect may be explained by a decrease in gaps between the epithelial cells, and increased expression of tight junctions. These findings and future studies could provide new therapeutic approaches for diseases affected by a compromised intestinal barrier function such as obesity, diabetes and NASH. Unless otherwise noted, all abstracts presented at ENDO are embargoed until the date and time of presentation. For oral presentations, the abstracts are embargoed until the session begins. s presented at a news conference are embargoed until the date and time of the news conference. The Endocrine Society reserves the right to lift the embargo on specific abstracts that are selected for promotion prior to or during ENDO.

Lipopolysaccharide at low concentration suppressed tight junction assembly in intestinal epithelial cells via AMPK suppression and MAPK pathway

Intestinal tight junction acts as a physical barrier and selective filter in intestine. Effect of low concentration of lipopolysaccharide (LPS) on AMPK activity, which is central to tight junction regulation, and its mechanism in intestinal epithelial cells (IEC) are not well characterized. This study aimed to investigate the effect of low‐concentration of LPS on AMPK suppression and intestinal tight junction permeability. To test whether low‐concentration of LPS inhibits AMPK phosphorylation, western blot analysis was performed. The result revealed that low dose LPS (1 ng/ml) suppresses AMPK activation in T84 cells. In order to investigate whether LPS suppressed intestinal tight junction assembly, calcium switch assays were performed. The result showed that LPS significantly suppressed tight junction assembly at 6 to 24 hours post‐treatment, indicating that low‐concentration of LPS inhibits tight junction assembly in IEC. Moreover, this effect was disappeared in the presence of U0126, a MAPK inhibitor. This data indicate that low‐concentration of LPS inhibits tight junction assembly via MAPK pathways. In conclusion, LPS induced AMPK suppression and tight junction permeability via MAPK pathway in IEC. This study shed light on the mechanism of intestinal diseases involving gram negative bacterial infection.Support or Funding InformationThis work is supported by Tonkla Ramathibodi program and Thailand Research Fund and Mahidol University. (BRG5980008)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Fructooligosaccharides induce tight junction assembly in intestinal epithelial cells via AMPK activation

Several intestinal and metabolic diseases including inflammatory bowel diseases (IBD), malnutrition, necrotizing enterocolitis and diabetes mellitus (DM) involve impairment of intestinal tight junction assembly. AMP‐activating protein kinase (AMPK) is an energy sensor that enhances tight junction assembly. Fructooligosaccharides (FOS) are well known prebiotics protecting against intestinal and metabolic diseases with unclear mechanisms. This study aimed to investigates effect of FOS on AMPK and tight junction assembly in intestinal epithelial cells (IEC). Using western blot analyses, it was found that FOS with concentrations from 0.001 to 10 mg/ml induced AMPK activation on T84 cells. FOS at concentration of 0.1 mg/ml showed the greatest effect on AMPK activation and it was used in the subsequent experiments. Calcium‐switch assays and fluorescein isothiocyanate (FITC) leakage experiments were then performed. The results revealed that FOS significantly increased percentage of TER at 15 minute, 1 hour, 6 hour, 12 hour and 24 hour post‐treatment. Interestingly, STO‐609, a CaMKKβ inhibitor, suppressed the effect of FOS on increasing TER and reducing FITC leakage. These results indicate that FOS increased tight junction assembly via CaMKKβ‐AMPK activation.Support or Funding InformationThis work is supported by Tonkla Ramathibodi program and Thailand Research Fund and Mahidol University (BRG5980008)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

A Myosin Chaperon, UNC‐45A, Is a Key Regulator of Intestinal Epithelial Apical Junctions

Epithelial adherens junctions (AJs) and tight junctions (TJs) play critical roles in regulating the integrity and permeability of epithelial barriers. Proper assembly and functioning of AJs and TJs depend on their associations with underlying actin filaments and the actin motor protein, nonmuscle myosin II (NM II). Our previous studies demonstrated the roles of NM II in controlling assembly and permeability of intestinal epithelial apical junctions in vitro and in vivo. However, the molecular mechanisms that regulate NM IIA functions in intestinal epithelium remain poorly understood. UNC‐45A is a highly conserved member of the UNC‐45/CRO1/She4p family proteins, which acts as a NM II chaperone and controls a proper folding and activity of myosin heavy chains. In the present study, we investigated the roles of UNC‐45A in the regulation of apical junctions in model intestinal epithelial cell monolayers.UNC‐45A protein was abundantly expressed in several commonly‐used human intestinal epithelial cell lines. Furthermore, its expression in T84 and HT‐29 cells was significantly downregulated by proinflammatory cytokines, IFN‐γ and TNF‐α, in parallel to cytokine induced TJ disassembly and barrier disruption. Consistently, UNC‐45A and NM IIA expression was downregulated in colonic epithelial cells isolated from tissue samples of ulcerative colitis patients. CRISPR/Cas9 mediated knock‐out of UNC‐45A in SK‐CO15 and HT‐29 human colonic epithelial cells resulted in multiple defects of apical junctions. These defects include the decreased transepithelial electrical resistance, increased transepithelial dextran flux, impaired steady‐state structure of AJs and TJs, and attenuated junctional reassembly during calcium switch. Together, our findings revealed a novel cytoskeleton‐dependent mechanism that regulates structure and functions of normal intestinal epithelial barrier and may contribute to barrier disruption during mucosal inflammation.Support or Funding InformationThis work was supported by NIH grant R01DK108278.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Palmitoylation of Claudin-5 Proteins Influences Their Lipid Domain Affinity and Tight Junction Assembly at the Blood-Brain Barrier Interface.

Post-translational lipid modification of integral membrane proteins is recognized as a key mechanism to modulate protein-protein and membrane-protein associations. Despite numerous reports of lipid-modified proteins, molecular-level understanding of the influence of lipid-modification of key membrane proteins remains elusive. This study focuses on the lipid modification of one such protein-claudin-5, a critical component of the blood-brain barrier tight junctions. Claudin-5 proteins are responsible for regulating the size and charge-selective permeability at the blood-brain interface. Palmitoylation of the claudin family of proteins is implicated in influencing the tight junction permeability in prior experimental studies. Here, we investigate the impact of palmitoylation on claudin-5 self-assembly using multiscale molecular simulations. To elucidate protein-membrane interactions, we used three model membrane compositions (endoplasmic reticulum, cholesterol-enriched endoplasmic reticulum, and plasma membrane) that mimic the complexity of cell organelles encountered by a typical membrane protein in its secretion pathway. The results show that palmitoylation enhances protein's affinity for cholesterol-rich domains in a membrane, and it can elicit a site-specific response based on the location of the palmitoyl chain on the protein. Also, in claudin-5 self-assembly, palmitoylation restricts specific protein-protein conformations. Overall, this study demonstrates the significance of post-translational lipid modification of proteins in cellular and subcellular membranes, and the impact palmitoylation can have on critical cellular functions of the protein.

Cytoplasmic polyadenylation element binding protein 2 (CPEB2) is required for tight-junction assembly for establishment of porcine trophectoderm epithelium.

Cytoplasmic polyadenylation element binding protein (CPEB) is an RNA-binding protein that promotes elongation of poly(A) tails and regulates mRNA translation. CPEB depletion in mammary epithelium is known to disrupt tight-junction (TJ) assembly via mislocalisation of tight junction protein 1 (TJP1), but the role of CPEB in the biological functions associated with TJs has not yet been studied. The objective of this study was to investigate the roles of CPEB2 during porcine parthenote development. CPEB2 was detected in both the nuclei and apical cytoplasm at the 4- and 8-cell stages and was localised to cell-cell contact after the initiation of the morula stage. Its depletion led to retarded blastocyst formation caused by impaired TJ assembly. Moreover, transcription of TJ-associated genes, including TJP1, Coxsackie virus and adenovirus receptor (CXADR) and occludin (OCLN), was not affected, but the corresponding proteins were not properly localised at the apical cell membrane in morulae, suggesting that CPEB2 confers mRNA stability or determines subcellular localisation for translation. Remarkably reduced relative levels of TJP1 transcripts bearing the 3'-untranslated region were noted, indicating that CPEB2 mediates TJP1 mRNA stability. In conclusion, our findings demonstrate that because of its regulation of TJP1, CPEB2 is required for TJ assembly during porcine blastocyst development.

Phase Separation of Zonula Occludens Proteins Drives Formation of Tight Junctions

Tight junctions are cell adhesion complexes that seal tissues and are involved in cell polarity and signalling. Supra-molecular assembly and positioning of tight junctions as continuous networks of adhesion strands is dependent on the two membrane associated scaffolding proteins ZO1 and ZO2. To understand how ZO proteins organize junction assembly, we performed quantitative cell biology and in vitro reconstitution experiments. We discovered that ZO proteins self-organize membrane attached compartments via phase separation. We identified the multivalent interactions of the conserved PDZ-SH3-GuK supra-domain as the driver of phase separation. These interactions are regulated by phosphorylation and intra-molecular binding. Formation of condensed ZO protein compartments is sufficient to specifically enrich and localize tight junction proteins including adhesion receptors, cytoskeletal adapters and transcription factors. Our results suggest that an active phase transition of ZO proteins into a condensed membrane bound compartment drives claudin polymerization and coalescence of a continuous tight junction belt.

An agonist of a zinc-sensing receptor GPR39 enhances tight junction assembly in intestinal epithelial cells via an AMPK-dependent mechanism

Intestinal barrier function depends on integrity of tight junctions, which serve as barriers to transepithelial influx of noxious substances/microorganisms from gut lumen. The G-protein coupled receptor 39 (GPR39) is a zinc-sensing receptor, which is expressed in several cell types including intestinal epithelial cells (IECs). The main objective of this study was to investigate the effect of GPR39 activation on tight junction assembly in IECs. Treatment with TC-G 1008 (1 μM –10 μM), a GPR39 agonist, and zinc (10 μM –100 μM) increased tight junction assembly in T84 cells. This effect was suppressed by pretreatment with compound C, an inhibitor of AMP-activated protein kinase (AMPK). In addition, western blot analysis revealed that treatment with TC-G 1008 induced AMPK activation in time- and concentration-dependent manners. Interestingly, inhibitors of phospholipase C (PLC) and calcium/calmodulin-dependent protein kinase kinase β (CaMKKβ) abrogated the effect of TC-G 1008 on inducing AMPK activation, tight junction assembly and zonula occludens-1 re-organization. Collectively, this study reveals a novel role of GPR39 in enhancing tight junction assembly in IECs via PLC-CaMKKβ-AMPK pathways. GPR39 agonists may be beneficial in the treatment of diseases associated impaired intestinal barrier function.

Anisomycin, a JNK and p38 activator, suppresses cell-cell junction formation in 2D cultures of K38 mouse keratinocyte cells and reduces claudin-7 expression, with an increase of paracellular permeability in 3D cultures.

Keratinocytes in the oral mucosal epithelium, which is a non-keratinized stratified epithelium, are exposed to various stimuli from the oral cavity. JNK and p38 are stress-activated mitogen-activated protein kinases (MAPKs) that are phosphorylated by various stimuli and are involved in the assembly and disassembly of tight junctions (TJs) in keratinocytes. Therefore, we investigated the effects of stress-activated MAPKs on TJs in a mouse keratinocyte cell line during cell-cell junction formation in two-dimensional (2D) cultures or stratification to form non-keratinized epithelium in 3D cultures. In 2D cultures, calcium induced zipper-like staining for ZO-1 at 2h and string-like staining for ZO-1 at 12h, which indicated immature and mature cell-cell junctions, respectively. Anisomycin (AM), a JNK and p38 activator, inhibited formation of string-like staining for ZO-1, whereas inhibition of JNK, but not p38, after AM treatment restored string-like staining for ZO-1, although claudins (CLDNs) 4, 6, and 7 did not completely colocalize to ZO-1-positive sites. In 3D cultures, AM treatment for 2 weeks activated only p38, suppressed flattening of the superficial cells, removed CLDN7 from ZO-1-positive spots on the surface of 3D cultures, which represent TJs, and decreased transepithelial electrical resistance. Thus, short-term AM treatment inhibited maturation of cell-cell junctions by JNK, but not p38, activation. p38 activation by long-term AM treatment affected morphology of stratified structures and paracellular permeability, which was increased by CLDN7 removal from TJs. Various chronic stimuli that activate stress-activated MAPKs may weaken the keratinocyte barrier and be involved in TJ-related diseases.