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Bacillus cereus is a Gram-positive bacterium that mainly causes self-limiting emetic or diarrheal illness but can also cause skin infections and bacteremia. Symptoms of B. cereus ingestion depend on the production of various toxins that target the gastric and intestinal epithelia. From a screen of bacterial isolates from human stool samples that compromised intestinal barrier function in mice, we identified a strain of B. cereus that disrupted tight and adherens junctions in the intestinal epithelium. This activity was mediated by the pore-forming exotoxin alveolysin, which increased the production of the membrane-anchored protein CD59 and of cilia- and flagella-associated protein 100 (CFAP100) in intestinal epithelial cells. In vitro, CFAP100 interacted with microtubules and promoted microtubule polymerization. CFAP100 overexpression stabilized microtubules in intestinal epithelial cells, leading to disorganization of the microtubule network and perturbation of tight and adherens junctions. The disruption of cell junctions by alveolysin depended on the increase in CFAP100, which in turn depended on CD59 and the activation of PI3K-AKT signaling. These findings demonstrate that, in addition to forming membrane pores, B. cereus alveolysin can permeabilize the intestinal epithelium by disrupting epithelial cell junctions in a manner that is consistent with intestinal symptoms and may allow the bacteria to escape the intestine and cause systemic infections. Our results suggest the potential value of targeting alveolysin or CFAP100 to prevent B. cereus–associated intestinal diseases and systemic infections. Toxin-induced junction dysfunction: The foodborne pathogen Bacillus cereus produces various toxins that damage intestinal epithelial cells. Sun et al. identified a strain of B. cereus that compromised the intestinal epithelial barrier in mice and permeabilized monolayers of cultured human colonic epithelial cells in a manner that depended on the secreted pore-forming toxin alveolysin. Although cells rapidly repaired acute membrane damage caused by alveolysin, the toxin also caused persistent disruption of cell junctions by increasing the production of the microtubule-binding protein CFAP100. CFAP100 promoted microtubule polymerization and stability, resulting in disorganization of the microtubule network and junction impairment. This mechanism likely contributes to the gastrointestinal symptoms of B. cereus infection and may enable the bacterium to escape the gut to cause systemic infections. -AMV [ABSTRACT FROM AUTHOR]

Epithelia maintain a functional barrier during tissue turnover while facing varying mechanical stress. This maintenance requires both dynamic cell rearrangements driven by actomyosin-linked intercellular adherens junctions and ability to adapt to and resist extrinsic mechanical forces enabled by keratin filament-linked desmosomes. How these two systems crosstalk to coordinate cellular movement and mechanical resilience is not known. Here we show that in stratifying epithelia the polarity protein aPKCλ controls the reorganization from stress fibers to cortical actomyosin during differentiation and upward movement of cells. Without aPKC, stress fibers are retained resulting in increased contractile prestress. This aberrant stress is counterbalanced by reorganization and bundling of keratins, thereby increasing mechanical resilience. Inhibiting contractility in aPKCλ−/− cells restores normal cortical keratin networks but also normalizes resilience. Consistently, increasing contractile stress is sufficient to induce keratin bundling and enhance resilience, mimicking aPKC loss. In conclusion, our data indicate that keratins sense the contractile stress state of stratified epithelia and balance increased contractility by mounting a protective response to maintain tissue integrity. [ABSTRACT FROM AUTHOR]

Apical-basal cell polarity must be tightly controlled for epithelial cyst and tubule formation, and these are important functional units in various epithelial organs. Polarization is achieved through the coordination of several molecules that divide cells into an apical domain and a basolateral domain, which are separated from tight and adherens junctions. Cdc42 regulates cytoskeletal organization and the tight junction protein ZO-1 at the apical margin of epithelial cell junctions. MST kinases control organ size through the regulation of cell proliferation and cell polarity. For example, MST1 relays the Rap1 signal to induce cell polarity and adhesion of lymphocytes. Our previous study showed that MST3 was involved in E-cadherin regulation and migration in MCF7 cells. In vivo, MST3 knockout mice exhibited higher ENaC expression at the apical site of renal tubules, resulting in hypertension. However, it was not clear whether MST3 was involved in cell polarity. Here, control MDCK cells, HA-MST3 and HA-MST3 kinase-dead (HA-MST3-KD) overexpressing MDCK cells were cultured in collagen or Matrigel. We found that the cysts of HA-MST3 cells were fewer and smaller than those of control MDCK cells; ZO-1 was delayed to the apical site of cysts and in cell-cell contact in the Ca2+ switch assay. However, HA-MST3-KD cells exhibited multilumen cysts. Intensive F-actin stress fibers were observed in HA-MST3 cells with higher Cdc42 activity; in contrast, HA-MST3-KD cells had lower Cdc42 activity and weaker F-actin staining. In this study, we identified a new MST3 function in the establishment of cell polarity through Cdc42 regulation. [ABSTRACT FROM AUTHOR]

The cell–cell connections in adherens junctions (AJs) are mediated by transmembrane receptors, type I cadherins (referred to here as cadherins). These cadherin-based connections (or trans bonds) are weak. To upregulate their strength, cadherins exploit avidity, the increased affinity of binding between cadherin clusters compared with isolated monomers. Formation of such clusters is a unique molecular process that is driven by a synergy of direct and indirect cis interactions between cadherins located at the same cell. In addition to their role in adhesion, cadherin clusters provide structural scaffolds for cytosolic proteins, which implicate cadherin into different cellular activities and signaling pathways. The cluster lifetime, which depends on the actin cytoskeleton, and on the mechanical forces it generates, determines the strength of AJs and their plasticity. The key aspects of cadherin adhesion, therefore, cannot be understood at the level of isolated cadherin molecules, but should be discussed in the context of cadherin clusters. Adherens junctions consist of cadherin clusters associated with distinct sets of cytosolic proteins. A synergy between two types of intercadherin interactions – trans (or adhesive) intercadherin interactions and indirect, through α-catenin, cadherin interactions with actin filaments – drives formation of cadherin clusters involved in cell–cell adhesion. The common theme of both interactions is their strengthening by pulling forces, which not only stabilize conformational changes of the corresponding binding domains, but also enhance linear (or cis) oligomerization of these domains. A repertoire of alternative clustering mechanisms appears to diversify intercadherin interactions forming a mosaic of functionally distinct clusters within a single adherens junction. [ABSTRACT FROM AUTHOR]

Cerebral cavernous malformations (CCMs) arise when capillaries within the brain enlarge abnormally, causing the blood–brain barrier (BBB) to break down. The BBB serves as a sophisticated interface that controls molecular interactions between the bloodstream and the central nervous system. The neurovascular unit (NVU) is a complex structure made up of neurons, astrocytes, endothelial cells (ECs), pericytes, microglia, and basement membranes, which work together to maintain blood–brain barrier (BBB) permeability. Within the NVU, tight junctions (TJs) and adherens junctions (AJs) between endothelial cells play a critical role in regulating the permeability of the BBB. Disruptions to these junctions can compromise the BBB, potentially leading to a hemorrhagic stroke. Understanding the molecular signaling cascades that regulate BBB permeability through EC junctions is, therefore, essential. New research has demonstrated that steroids, including estrogens (ESTs), glucocorticoids (GCs), and metabolites/derivatives of progesterone (PRGs), have multifaceted effects on blood–brain barrier (BBB) permeability by regulating the expression of tight junctions (TJs) and adherens junctions (AJs). They also have anti-inflammatory effects on blood vessels. PRGs, in particular, have been found to play a significant role in maintaining BBB integrity. PRGs act through a combination of its classic and non-classic PRG receptors (nPR/mPR), which are part of a signaling network known as the CCM signaling complex (CSC). This network couples both nPR and mPR in the CmPn/CmP pathway in endothelial cells (ECs). [ABSTRACT FROM AUTHOR]

Metastasis-suppressor 1 (MTSS1) is a membrane-interacting scaffolding protein that regulates the integrity of epithelial cell-cell junctions and functions as a tumor suppressor in a wide range of carcinomas. MTSS1 binds phosphoinositide-rich membranes through its I-BAR domain and is capable of sensing and generating negative membrane curvature in vitro. However, the mechanisms by which MTSS1 localizes to intercellular junctions in epithelial cells and contributes to their integrity and maintenance have remained elusive. By carrying out EM and live-cell imaging on cultured Madin-Darby canine kidney cell monolayers, we provide evidence that adherens junctions of epithelial cells harbor lamellipodia-like, dynamic actin-driven membrane folds, which exhibit high negative membrane curvature at their distal edges. BioID proteomics and imaging experiments demonstrated that MTSS1 associates with an Arp2/3 complex activator, the WAVE-2 complex, in dynamic actin-rich protrusions at cell-cell junctions. Inhibition of Arp2/3 or WAVE-2 suppressed actin filament assembly at adherens junctions, decreased the dynamics of junctional membrane protrusions, and led to defects in epithelial integrity. Together, these results support a model in which membrane-associated MTSS1, together with the WAVE-2 and Arp2/3 complexes, promotes the formation of dynamic lamellipodia-like actin protrusions that contribute to the integrity of cell-cell junctions in epithelial monolayers. [ABSTRACT FROM AUTHOR]

Testicular development and spermatogenesis are tightly regulated by the number of genes and noncoding genes, and mRNAs and lncRNAs play vital roles in regulating posttranscriptional gene expression. However, mRNAs and lncRNAs have not been systematically identified in the testes of donkeys. In this study, mRNA and lncRNA expression profiles in the testes of DeZhou donkeys between 2 months and 2 years of age were comprehensively analysed by RNA sequencing. We identified 56,605 lncRNAs and 61,857 mRNAs by gene expression analysis, and 21,845 lncRNAs (p <.05) and 14,109 mRNAs (p <.05) were differentially expressed in the immature (2‐month‐old, n = 3, noADGW) and mature (2‐year‐old, n = 3, ADGW) stages. In addition, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses revealed that the predicted target genes were enriched in the adherens junction, cell cycle, propanoate metabolism and cell adhesion molecule pathways. This study identified and analysed a comprehensive catalogue of lncRNAs and mRNAs in donkey testes, which provides a useful resource for further investigation of biological function in donkey lncRNAs. [ABSTRACT FROM AUTHOR]

In vitro vascular wall bilayer models for drug testing and disease modeling must emulate the physical and biological properties of healthy vascular tissue and its endothelial barrier function. Both endothelial cell (EC)‐vascular smooth muscle cell (SMC) interaction across the internal elastic lamina (IEL) and blood vessel stiffness impact endothelial barrier integrity. Polymeric porous track‐etched membranes (TEM) typically represent the IEL in laboratory vascular bilayer models. However, TEM stiffness exceeds that of diseased blood vessels, and the membrane pore architecture limits EC‐SMC interaction. The mechanical properties of compliant honeycomb film (HCF) membranes better simulate the Young's modulus of healthy blood vessels, and HCFs are thinner (4 vs. 10 μm) and more porous (57 vs. 6.5%) than TEMs. We compared endothelial barrier integrity in vascular wall bilayer models with human ECs and SMCs statically cultured on opposite sides of HCFs and TEMs (5 μm pores) for up to 12 days. Highly segregated localization of tight junction (ZO‐1) and adherens junction (VE‐cadherin) proteins and quiescent F‐actin cytoskeletons demonstrated superior and earlier maturation of interendothelial junctions. Quantifying barrier integrity based on transendothelial electrical resistance (TEER), membranes showed only minor but significant TEER differences despite enhanced junctional protein localization on HCF. Elongated ECs on HCF likely experienced greater paracellular diffusion than blocky ECs on TEM. Also, larger populations of plaques of connexin 43 subunit‐containing gap junctions suggested enhanced EC‐SMC communication across the more porous, thinner HCF. Compared with standard TEMs, engineered vascular wall bilayers cultured on HCFs better replicate physiologic endothelial barrier integrity. [ABSTRACT FROM AUTHOR]

Three-dimensional cell culture models are increasingly adopted as preferred pre-clinical drug testing platforms, as they circumvent limitations associated with traditional monolayer cell cultures. However, many of these models are not fully characterized. This study aimed to characterize a BT-20 triple-negative breast carcinoma spheroid model and assess its susceptibility to doxorubicin in comparison to a monolayer model. Spheroids were developed using the liquid overlay method. Phenotypic attributes were analyzed by characterizing changes in size, gross morphology, protein content, metabolic activity, hypoxic status, and cell–cell junctions. The cytotoxic range of doxorubicin in monolayers was determined using the sulforhodamine B assay, and the comparative effect of toxic and sub-toxic concentrations was assessed in both spheroids and monolayers. Similar to the in vivo microenvironment, spheroids had a heterogeneous spatial cytoarchitecture, inherent hypoxia and strong adherens junctions. Doxorubicin induced dose-dependent cytotoxicity in monolayers (IC25: 130 nM, IC50: 320 nM and IC75: 1580 nM); however, these concentrations did not alter the spheroid size or acid phosphatase activity. Only concentrations ≥6 µM had any effect on spheroid integrity. In comparison to monolayers, the BT-20 spheroid model has decreased sensitivity to doxorubicin and could serve as a better model for susceptibility testing in triple-negative breast cancer. [ABSTRACT FROM AUTHOR]

Chronic nonbacterial osteomyelitis (CNO) is an autoinflammatory bone disorder. The origin and development of CNO involve many complex immune processes, resulting in delayed diagnosis and a lack of effective treatment. Although bioinformatics analysis has been utilized to seek key genes and pathways in CNO, only a few bioinformatics studies that focus on CNO pathogenesis and mechanisms have been reported. This study aimed to identify key biomarkers that could serve as early diagnostic or therapeutic markers for CNO. Two RNA-seq datasets (GSE133378 and GSE187429) were obtained from the Gene Expression Omnibus (GEO). Weighted gene coexpression network analysis (WGCNA) and differentially expressed gene (DEG) analysis were conducted to identify the genes associated with CNO. Then, the autoinflammatory genes most associated with CNO were identified based on the GeneCards database and a CNO prediction model, which was created by the LASSO machine learning algorithm. The accuracy of the model and effects of the autoinflammatory genes according to receiver operating characteristic (ROC) curves were verified in external datasets (GSE7014). Finally, we performed clustering analysis with ConsensusClusterPlus. In total, eighty CNO-related genes were identified and were significantly enriched in the biological processes regulation of actin filament organization, cell–cell junction organization and gamma-catenin binding. The main enriched pathways were adherens junctions, viral carcinogenesis and systemic lupus erythematosus. Two autoinflammatory genes with high expression in CNO samples were identified by combining an optimal machine learning algorithm (LASSO) with the GeneCards database. An external validation dataset (GSE187429) was utilized for ROC analysis of the prediction model and two genes, and the results indicated good efficiency. Then, based on consensus clustering analysis, we found that the expression of UTS2 and MPO differed between clusters. Finally, the ceRNA network of lncRNAs and the small molecule compounds targeting the two autoinflammatory genes were predicted. The identification of two autoinflammatory genes, the HCG18/has-mir-147a/UTS2/MPO axis and signalling pathways in this study can help us understand the molecular mechanism of CNO formation and provides candidate targets for the diagnosis and treatment of CNO. [ABSTRACT FROM AUTHOR]

The oral cavity is directly exposed to a variety of environmental stimuli and contains a diverse microbiome that continuously interacts with the oral epithelium. Therefore, establishment and maintenance of the barrier function of the oral mucosa is of paramount importance for its function and for the body's overall health. The adherens junction is a cell–cell adhesion complex that is essential for epithelial barrier function. Although a considerable body of work has associated barrier disruption with oral diseases, the molecular underpinnings of these associations have not been equally investigated. This is critical, since adherens junction components also possess significant signaling roles in the cell, in addition to their architectural ones. Here, we summarize current knowledge involving adherens junction components in oral pathologies, such as cancer and oral pathogen-related diseases, while we also discuss gaps in the knowledge and opportunities for future investigation of the relationship between adherens junctions and oral diseases. [ABSTRACT FROM AUTHOR]

Since nanoparticles are taken up into cells by endocytosis, phagocytosis, or pinocytosis, they have been studied as intracellular drug carriers. Janus particles have an anisotropic structure composed of two or more distinct domains and have been proposed for use in various applications, including use as imaging agents or nanosensors. This study aimed to clarify the influence of the type of nanoparticles on their distribution in a human Caucasian colon adenocarcinoma (Caco-2) cell monolayer. We fabricated Janus and conventional spherical nanoparticles composed of pharmaceutically applicable ingredients. Janus and spherical nanoparticles composed of a cationic polymer and surfactant lipids were prepared by controlling the solvent removal pattern from the oil phase in the solvent removal process using the solvent evaporation and solvent diffusion methods. The distribution of nanoparticles in the Caco-2 cell monolayer was then evaluated using confocal laser microscopy. The mean hydrodynamic size of the fabricated Janus nanoparticles was 119.2 ± 4.6 nm. Distribution analysis using Caco-2 cells suggested that Janus nanoparticles were localized around the adherens junctions located just below the tight junction. Clear localization was not observed in non-Janus nanoparticles with the same composition. The clear localization of the Janus nanoparticles around the adherens junction may be due to their positive charge and asymmetric structure. Our results suggest the considerable potential for the development of nanoparticulate drug carriers to target cellular gaps. [ABSTRACT FROM AUTHOR]

Simple Summary: Nasopharyngeal carcinoma (NPC) is endemic to southern China and cancer metastasis remains the main cause of treatment failures. Previously we have found THY1, a cell surface glycoprotein, inhibits the invasion of NPC cells and functions as a tumor suppressor in NPC. In the present study, we further illustrated the mechanism that contributes to the tumor suppressive function of THY1. Two binding partners of THY1, PDGF-Rβ and PTPN22, were identified, and PTPN22 represents the downstream signaling molecule of THY1 to inhibit the PDGF-Rβ–induced SRC activity. The anti-metastatic effect of SRC inhibition was subsequently validated in a mice model with administration of a SRC inhibitor that has been used in clinics for other diseases. This study opens a new window to target the SRC signaling activity which is antagonized by THY1 in NPC. We had previously shown that THY1 (CD90) is a tumor suppressor in nasopharyngeal carcinoma (NPC) and that its down-regulation and loss of expression are associated with tumor metastasis, yet the mechanism leading to such effects remains unknown. In this study we show that tumor invasion could be suppressed by THY1 via adherens junction formation in a few NPC cell lines, and knockdown of THY1 would disrupt this cell-cell adhesion phenotype. Mechanistically, the activity of the SRC family kinase (SFK) member, SRC, and canonical Wnt signaling were dramatically reduced when THY1 was constitutively expressed. Previous studies by others have found that high levels of SRC activity in NPCs are associated with EMT and a poor prognosis. We hypothesized that THY1 can suppress tumor invasion in NPC via inhibition of SRC. By gene silencing of SRC, we found that the in vitro NPC cell invasion was significantly reduced and adherens junctions were restored. Through proteomic analysis, we identified that platelet-derived growth factor receptor β (PDGF-Rβ) and protein tyrosine phosphatase nonreceptor type 22 (PTPN22) are novel and potential binding partners of THY1, which were subsequently verified by co-immunoprecipitation (co-IP) analysis. The ligand of PDGF-Rβ (PDGF-BB) could highly induce SRC activation and NPC cell invasion, which could be almost completely suppressed by THY1 expression. On the other hand, the PTPN22 siRNA could enhance both the SRC activities and the cell invasion and could also disrupt the adherens junctions in the THY1-expressing NPC cells; the original THY1-induced phenotypes were reverted when the PTPN22 expression was reduced. Together, our results identified that PTPN22 is essential for THY1 to suppress cell invasion and SRC activity, maintain tight adherens junctions, and prevent NPC metastasis. These results suggested that PDGF-Rβ and SRC can be used as drug targets for suppressing NPC metastasis. Indeed, our in vivo assay using the SRC inhibitor KX2-391, clearly showed that inhibition of SRC signaling can prevent the metastasis of NPC, indicating that targeting SRC can be a promising approach to control the NPC progression. [ABSTRACT FROM AUTHOR]

Epithelial to Mesenchymal Transition (EMT) is a dynamic, morphogenetic process characterized by a phenotypic shift in epithelial cells towards a motile and often invasive mesenchymal phenotype. We have previously demonstrated that EMT is associated with an increase in assembly of the extracellular matrix protein fibronectin (FN) into insoluble, viscoelastic fibrils. We have also demonstrated that Transforming Growth Factor- β 1 (TGF- β 1) localizes to FN fibrils, and disruption of FN assembly or disruption of TGF- β 1 localization to FN fibrils attenuates EMT. Previous studies have shown that TGF- β 1 induces spatial gradients of EMT in mammary epithelial cells cultured on FN islands, with cells at free edges of the island preferentially undergoing EMT. In the current work, we sought to investigate: (a) whether FN fibril assembly is also spatially patterned in response to TGF- β 1, and (b) what effects FN fibril inhibition has on spatial gradients of E-Cadherin and FN fibrillogenesis. We demonstrate that mammary epithelial cells cultured on square micropatterns have fewer E-Cadherin-containing adherens junctions and assemble more FN fibrils at the periphery of the micropattern in response to increasing TGF- β 1 concentration, indicating that TGF- β 1 induces a spatial gradient of both E-Cadherin and FN fibrils. Inhibition of FN fibril assembly globally diminished E-Cadherin-containing adherens junctions and FN fibrillogenesis, but did not eliminate the spatial gradient of either. This suggests that global inhibition of FN reduces the degree of both FN fibrillogenesis and E-Cadherin-containing adherens junctions, but does not eliminate the spatial gradient of either, suggesting that spatial gradients of EMT and FN fibrillogenesis are influenced by additional factors. [ABSTRACT FROM AUTHOR]

Chemotherapy- or inflammation-induced increase in intestinal permeability represents a severe element in disease evolution in patients suffering from colorectal cancer and gut inflammatory conditions. Emerging data strongly support the gut microbiota's role in preserving intestinal barrier integrity, whilst both chemotherapy and gut inflammation alter microbiota composition. Some probiotics might have a strong re-balancing effect on the gut microbiota, also positively affecting intestinal barrier integrity. In this study, we asked whether Limosilactobacillus fermentum ME-3 can prevent the intestinal paracellular permeability increase caused by the chemotherapeutic drug Irinotecan or by inflammatory stimuli, such as lipopolysaccharide (LPS). As an intestinal barrier model, we used a confluent and polarized Caco-2 cell monolayer and assessed the ME-3-induced effect on paracellular permeability by transepithelial electrical resistance (TEER) and fluorescent-dextran flux assays. The integrity of tight and adherens junctions was examined by confocal microscopy analysis. Transwell co-cultures of Caco-2 cells and U937-derived macrophages were used as models of LPS-induced intestinal inflammation to test the effect of ME-3 on release of the pro-inflammatory cytokines Tumor Necrosis Factor α, Interleukin-6, and Interleukin-8, was measured by ELISA. The results demonstrate that ME-3 prevents the IRI-induced increment in paracellular permeability, possibly by modulating the expression and localization of cell junction components. In addition, ME-3 inhibited both the increase in paracellular permeability and the release of pro-inflammatory cytokines in the co-culture model of LPS-induced inflammation. Our findings sustain the validity of L. fermentum ME-3 as a valuable therapeutic tool for preventing leaky gut syndrome, still currently without an available specific treatment. [ABSTRACT FROM AUTHOR]

Two prominent concepts for the sensing of shear stress by endothelium are the PIEZO1 channel as a mediator of mechanically activated calcium ion entry and the PECAM1 cell adhesion molecule as the apex of a triad with CDH5 and VGFR2. Here, we investigated if there is a relationship. By inserting a non-disruptive tag in native PIEZO1 of mice, we reveal in situ overlap of PIEZO1 with PECAM1. Through reconstitution and high resolution microscopy studies we show that PECAM1 interacts with PIEZO1 and directs it to cell-cell junctions. PECAM1 extracellular N-terminus is critical in this, but a C-terminal intracellular domain linked to shear stress also contributes. CDH5 similarly drives PIEZO1 to junctions but unlike PECAM1 its interaction with PIEZO1 is dynamic, increasing with shear stress. PIEZO1 does not interact with VGFR2. PIEZO1 is required in Ca2+-dependent formation of adherens junctions and associated cytoskeleton, consistent with it conferring force-dependent Ca2+ entry for junctional remodelling. The data suggest a pool of PIEZO1 at cell junctions, the coming together of PIEZO1 and PECAM1 mechanisms and intimate cooperation of PIEZO1 and adhesion molecules in tailoring junctional structure to mechanical requirement. The PIEZO1 channel and the PECAM1 cell adhesion molecule interact and PIEZO1 cooperates with PECAM1 and CDH5 cell junctions during mechanical stress. [ABSTRACT FROM AUTHOR]

Proteins and peptides often require frequent needle-based administrations. Here, we report a non-parenteral delivery method for proteins through physical mixing with protamine, an FDA-approved peptide. Protamine was shown to promote tubulation and rearrangement of cellular actin, leading to enhanced intracellular delivery of proteins compared to poly(arginine) 8 (R8). While the R8-mediated delivery resulted in significant lysosomal accumulation of the cargo, protamine directed the proteins to the nuclei with little lysosomal uptake. Intranasal delivery of insulin mixed with protamine effectively reduced blood glucose levels in diabetic mice 0.5 h after administration and the effect lasted for ∼6 h, comparable to subcutaneously injected insulin at the same dose. In mice, protamine was shown to overcome mucosal and epithelial barriers and modulate adherens junctions, promoting insulin penetration to the lamina propria layer for systemic absorption. [Display omitted] • Protamine increased intracellular delivery of proteins compared to R8 in 2D and 3D cell models. • Proteins delivered by protamine displayed cytosolic delivery and little accumulation in lysosomes. • Protamine enhanced intracellular delivery of proteins by promoting actin rearrangement of tubulation. • Protamine increased systemic absorption of proteins via intranasal delivery compared to R8. [ABSTRACT FROM AUTHOR]

The integrity and permeability of epithelial and endothelial barriers depend on the formation of tight junctions, adherens junctions, and a junction-associated cytoskeleton. The establishment of this junction–cytoskeletal module relies on the correct folding and oligomerization of its protein components. Molecular chaperones are known regulators of protein folding and complex formation in different cellular compartments. Mammalian cells possess an elaborate chaperone network consisting of several hundred chaperones and co-chaperones. Only a small part of this network has been linked, however, to the regulation of intercellular adhesions, and the systematic analysis of chaperone functions at epithelial and endothelial barriers is lacking. This review describes the functions and mechanisms of the chaperone-assisted regulation of intercellular junctions. The major focus of this review is on heat shock protein chaperones, their co-chaperones, and chaperonins since these molecules are the focus of the majority of the articles published on the chaperone-mediated control of tissue barriers. This review discusses the roles of chaperones in the regulation of the steady-state integrity of epithelial and vascular barriers as well as the disruption of these barriers by pathogenic factors and extracellular stressors. Since cytoskeletal coupling is essential for junctional integrity and remodeling, chaperone-assisted assembly of the actomyosin cytoskeleton is also discussed.

Multifunctional magnetic nanoparticles have shown great promise as next-generation imaging and perturbation probes for deciphering molecular and cellular processes. As a consequence of multicomponent integration into a single nanosystem, pre-existing nanoprobes are typically large and show limited access to biological targets present in a crowded microenvironment. Here, we apply organic-phase surface PEGylation, click chemistry, and charge-based valency discrimination principles to develop compact, modular, and monovalent magnetofluorescent nanoparticles (MFNs). We show that MFNs exhibit highly efficient labeling to target receptors present in cells with a dense and thick glycocalyx layer. We use these MFNs to interrogate the E-cadherin-mediated adherens junction formation and F-actin polymerization in a three-dimensional space, demonstrating the utility as modular and versatile mechanogenetic probes in the most demanding single-cell perturbation applications.

Arrhythmogenic cardiomyopathy (ACM) is an inherited progressive cardiac disease. Many patients with ACM harbor mutations in desmosomal genes, predominantly in plakophilin-2 (PKP2). Although the genetic basis of ACM is well characterized, the underlying disease-driving mechanisms remain unresolved. Explanted hearts from patients with ACM had less PKP2 compared with healthy hearts, which correlated with reduced expression of desmosomal and adherens junction (AJ) proteins. These proteins were also disorganized in areas of fibrotic remodeling. In vitro data from human-induced pluripotent stem cell–derived cardiomyocytes and microtissues carrying the heterozygous PKP2 c.2013delC pathogenic mutation also displayed impaired contractility. Knockin mice carrying the equivalent heterozygous Pkp2 c.1755delA mutation recapitulated changes in desmosomal and AJ proteins and displayed cardiac dysfunction and fibrosis with age. Global proteomics analysis of 4-month-old heterozygous Pkp2 c.1755delA hearts indicated involvement of the ubiquitin-proteasome system (UPS) in ACM pathogenesis. Inhibition of the UPS in mutant mice increased area composita proteins and improved calcium dynamics in isolated cardiomyocytes. Additional proteomics analyses identified lysine ubiquitination sites on the desmosomal proteins, which were more ubiquitinated in mutant mice. In summary, we show that a plakophilin-2 mutation can lead to decreased desmosomal and AJ protein expression through a UPS-dependent mechanism, which preceded cardiac remodeling. These findings suggest that targeting protein degradation and improving desmosomal protein stability may be a potential therapeutic strategy for the treatment of ACM. PKPing up the rhythm: Arrhythmogenic cardiomyopathy (ACM) is a debilitating disease with clear genetic links but variable penetrance and poorly understood mechanisms. Here Tsui and colleagues analyzed heart tissue from patients with ACM along with inducible pluripotent stem cell–derived cardiomyocytes and mice carrying a plakophilin-2 (Pkp2) mutation to reveal that decreased expression of desmosomal and adherens junction proteins correlated with cardiac dysfunction and fibrosis. Proteomics data indicated involvement of the ubiquitin-proteasome system (UPS) in the degradation of these proteins, and inhibition of the UPS improved protein expression and calcium dynamics in isolated mutant cardiomyocytes. These results suggest that therapies aimed at increasing stability of desmosomal proteins may improve cardiac function in patients with ACM.--AW [ABSTRACT FROM AUTHOR]

Introduction: The movement of fluids and solutes across the ependymal barrier, and their changes in physiologic and disease states are poorly understood. This gap in knowledge contributes strongly to treatment failures and complications in various neurological disorders. Methods: We systematically searched and reviewed original research articles treating ependymal intercellular junctions on PubMed. Reviews, opinion papers, and abstracts were excluded. Research conducted on tissue samples, cell lines, CSF, and animal models was considered. Results: A total of 45 novel articles treating tight, adherens and gap junctions of the ependyma were included in our review, spanning from 1960 to 2022. The findings of this review point toward a central and not yet fully characterized role of the ependymal lining ultrastructure in fluid flow interactions in the brain. In particular, tight junctions circumferentially line the apical equator of ependymal cells, changing between embryonal and adult life in several rodent models, shaping fluid and solute transit in this location. Further, adherens and gap junctions appear to have a pivotal role in several forms of congenital hydrocephalus. Conclusions: These findings may provide an opportunity for medical management of CSF disorders, potentially allowing for tuning of CSF secretion and absorption. Beyond hydrocephalus, stroke, trauma, this information has relevance for metabolite clearance and drug delivery, with potential to affect many patients with a variety of neurological disorders. This critical look at intercellular junctions in ependyma and the surrounding interstitial spaces is meant to inspire future research on a central and rather unknown component of the CSF-brain interface. [ABSTRACT FROM AUTHOR]

Multidirectional or disturbed flow promotes endothelial dysfunction and is associated with early atherogenesis. Here we investigated the role of Wnt signalling in flow-mediated endothelial dysfunction. The expression of Frizzled-4 was higher in cultured human aortic endothelial cells (ECs) exposed to disturbed flow compared to that seen for undisturbed flow, obtained using an orbital shaker. Increased expression was also detected in regions of the porcine aortic arch exposed to disturbed flow. The increased Frizzled-4 expression in cultured ECs was abrogated following knockdown of R-spondin-3. Disturbed flow also increased the nuclear localisation and activation of ß-catenin, an effect that was dependent on Frizzled-4 and Rspondin-3. Inhibition of ß-catenin using the small-molecule inhibitor iCRT5 or knockdown of Frizzled-4 or R-spondin-3 resulted in reduced expression of pro-inflammatory genes in ECs exposed to disturbed flow, as did inhibition of WNT5A signalling. Inhibition of the canonical Wnt pathway had no effect. Inhibition of ß-catenin also reduced endothelial paracellular permeability; this was associated with altered junctional and focal adhesion organisation and cytoskeletal remodelling. These data suggest the presence of an atypical Frizzled-4-ß-catenin pathway that promotes endothelial dysfunction in response to disturbed flow. [ABSTRACT FROM AUTHOR]

Cancers, such as squamous cell carcinoma, frequently invade as multicellular units. However, these invading units can be organised in a variety of ways, ranging from thin discontinuous strands to thick 'pushing' collectives. Here we employ an integrated experimental and computational approach to identify the factors that determine the mode of collective cancer cell invasion. We find that matrix proteolysis is linked to the formation of wide strands but has little effect on the maximum extent of invasion. Cell-cell junctions also favour wide strands, but our analysis also reveals a requirement for cell-cell junctions for efficient invasion in response to uniform directional cues. Unexpectedly, the ability to generate wide invasive strands is coupled to the ability to grow effectively when surrounded by extracellular matrix in three-dimensional assays. Combinatorial perturbation of both matrix proteolysis and cell-cell adhesion demonstrates that the most aggressive cancer behaviour, both in terms of invasion and growth, is achieved at high levels of cell-cell adhesion and high levels of proteolysis. Contrary to expectation, cells with canonical mesenchymal traits - no cell-cell junctions and high proteolysis - exhibit reduced growth and lymph node metastasis. Thus, we conclude that the ability of squamous cell carcinoma cells to invade effectively is also linked to their ability to generate space for proliferation in confined contexts. These data provide an explanation for the apparent advantage of retaining cell-cell junctions in squamous cell carcinomas. [ABSTRACT FROM AUTHOR]

Adherens junctions (AJs) are a defining feature of all epithelial cells. They regulate epithelial tissue architecture and integrity, and their dysregulation is a key step in tumor metastasis. AJ remodeling is crucial for cancer progression, and it plays a key role in tumor cell survival, growth, and dissemination. Few studies have examined AJ remodeling in cancer cells consequently, it remains poorly understood and unleveraged in the treatment of metastatic carcinomas. Fascin1 is an actin‐bundling protein that is absent from the normal epithelium but its expression in colon cancer is linked to metastasis and increased mortality. Here, we provide the molecular mechanism of AJ remodeling in colon cancer cells and identify for the first time, fascin1's function in AJ remodeling. We show that in colon cancer cells fascin1 remodels junctional actin and actomyosin contractility which makes AJs less stable but more dynamic. By remodeling AJs fascin1 drives mechanoactivation of WNT/β‐catenin signaling and generates "collective plasticity" which influences the behavior of cells during cell migration. The impact of mechanical inputs on WNT/β‐catenin activation in cancer cells remains poorly understood. Our findings highlight the role of AJ remodeling and mechanosensitive WNT/β‐catenin signaling in the growth and dissemination of colorectal carcinomas. [ABSTRACT FROM AUTHOR]

Significance: KRIT1 (Krev interaction trapped 1) is a scaffolding protein that plays a critical role in vascular morphogenesis and homeostasis. Its loss-of-function has been unequivocally associated with the pathogenesis of Cerebral Cavernous Malformation (CCM), a major cerebrovascular disease of genetic origin characterized by defective endothelial cell–cell adhesion and ensuing structural alterations and hyperpermeability in brain capillaries. KRIT1 contributes to the maintenance of endothelial barrier function by stabilizing the integrity of adherens junctions and inhibiting the formation of actin stress fibers. Recent Advances: Among the multiple regulatory mechanisms proposed so far, significant evidence accumulated over the past decade has clearly shown that the role of KRIT1 in the stability of endothelial barriers, including the blood-brain barrier, is largely based on its involvement in the complex machinery governing cellular redox homeostasis and responses to oxidative stress and inflammation. KRIT1 loss-of-function has, indeed, been demonstrated to cause an impairment of major redox-sensitive mechanisms involved in spatiotemporal regulation of cell adhesion and signaling, which ultimately leads to decreased cell–cell junction stability and enhanced sensitivity to oxidative stress and inflammation. Critical Issues: This review explores the redox mechanisms that influence endothelial cell adhesion and barrier function, focusing on the role of KRIT1 in such mechanisms. We propose that this supports a novel model wherein redox signaling forms the common link between the various pathogenetic mechanisms and therapeutic approaches hitherto associated with CCM disease. Future Directions: A comprehensive characterization of the role of KRIT1 in redox control of endothelial barrier physiology and defense against oxy-inflammatory insults will provide valuable insights into the development of precision medicine strategies. Antioxid. Redox Signal. 38, 496–528. [ABSTRACT FROM AUTHOR]

Angiogenesis, neovascularization, and vascular remodeling are highly dynamic processes, where endothelial cell–cell adhesion within the vessel wall controls a range of physiological processes, such as growth, integrity, and barrier function. The cadherin–catenin adhesion complex is a key contributor to inner blood–retinal barrier (iBRB) integrity and dynamic cell movements. However, the pre-eminent role of cadherins and their associated catenins in iBRB structure and function is not fully understood. Using a murine model of oxygen-induced retinopathy (OIR) and human retinal microvascular endothelial cells (HRMVECs), we try to understand the significance of IL-33 on retinal endothelial barrier disruption, leading to abnormal angiogenesis and enhanced vascular permeability. Using electric cell-substrate impedance sensing (ECIS) analysis and FITC-dextran permeability assay, we observed that IL-33 at a 20 ng/mL concentration induced endothelial-barrier disruption in HRMVECs. The adherens junction (AJs) proteins play a prominent role in the selective diffusion of molecules from the blood to the retina and in maintaining retinal homeostasis. Therefore, we looked for the involvement of adherens junction proteins in IL-33-mediated endothelial dysfunction. We observed that IL-33 induces α-catenin phosphorylation at serine/threonine (Ser/Thr) residues in HRMVECs. Furthermore, mass-spectroscopy (MS) analysis revealed that IL-33 induces the phosphorylation of α-catenin at Thr654 residue in HRMVECs. We also observed that PKCμ/PRKD1-p38 MAPK signaling regulates IL-33-induced α-catenin phosphorylation and retinal endothelial cell-barrier integrity. Our OIR studies revealed that genetic deletion of IL-33 resulted in reduced vascular leakage in the hypoxic retina. We also observed that the genetic deletion of IL-33 reduced OIR-induced PKCμ/PRKD1-p38 MAPK-α-catenin signaling in the hypoxic retina. Therefore, we conclude that IL-33-induced PKCμ/PRKD1-p38 MAPK-α-catenin signaling plays a significant role in endothelial permeability and iBRB integrity. [ABSTRACT FROM AUTHOR]

Hemagglutinin (HA), a nontoxic component of the botulinum neurotoxin (BoNT) complex, binds to E-cadherin and inhibits E-cadherin-mediated cell-cell adhesion.HAis a 470 kDa protein complex comprising six HA1, three HA2, and three HA3 subcomponents. Thus, to prepare recombinant full-length HA in vitro, it is necessary to reconstitute the macromolecular complex from purified HA subcomponents, which involves multiple purification steps. In this study, we developed NanoHA, a minimal E-cadherin inhibitor protein derived from Clostridium botulinum HA with a simple purification strategy needed for production. NanoHA, containing HA2 and a truncated mutant of HA3 (amino acids 380-626; termed as HA3mini), is a 47 kDa single polypeptide (one-tenth the molecular weight of full-length HA, 470 kDa) engineered with three types of modifications: (i) a short linker sequence between the C terminus of HA2 and N terminus of HA3; (ii) a chimeric complex composed of HA2 derived from the serotype C BoNT complex and HA3mini from the serotype B BoNT complex; and (iii) three amino acid substitutions from hydrophobic to hydrophilic residues on the protein surface. We demonstrated that NanoHA inhibits E-cadherin-mediated cell-cell adhesion of epithelial cells (e.g., Caco-2 and Madin-Darby canine kidney cells) and disrupts their epithelial barrier. Finally, unlike full-length HA, NanoHA can be transported from the basolateral side to adherens junctions via passive diffusion. Overall, these results indicate that the rational design of NanoHA provides a minimal E-cadherin inhibitor with a wide variety of applications as a lead molecule and for further molecular engineering. [ABSTRACT FROM AUTHOR]

Background. FGF signaling is critical to controlling various cancers. Nevertheless, the functions of FGF-related genes in PCa are still unknown. Objective. The objective of this study is to build a FGF-related signature that was capable of accurately predicting PCa survival and prognosis for BCR. Methods. The univariate and multivariate Cox regression, infiltrating immune cells, LASSO, and GSEA analyses were carried out to build a prognostic model. Results. A FGF-related signature that consists of PIK3CA and SOS1 was developed for the purpose of predicting PCa prognosis, and all patients were categorized into low- and high-risk groups. In comparison to the low-risk group, high-risk score patients had poorer BCR survival. This signature's predictive power has been investigated utilizing the AUC of the ROC curves. The risk score has been shown to be an independent prognostic factor by multivariate analysis. The four enriched pathways of the high-risk group were obtained by gene set enrichment analysis (GSEA) and found to be associated with the tumorigenesis and development of PCa, including focal adhesion, TGF-β signaling pathway, adherens junction, and ECM receptor interaction. The high-risk groups had considerably higher levels of immune status and tumor immune cell infiltration, suggesting a more favorable response to immune checkpoint inhibitors. IHC found that the expression of the two FGF-related genes in the predictive signature was extremely different in PCa tissues. Conclusion. To summarize, our FGF-related risk signature may effectively predict and diagnose PCa, indicating that in PCa patients, they are potential therapeutic targets and promising prognostic biomarkers. [ABSTRACT FROM AUTHOR]

The endothelial cells (EC) of established blood vessels in adults remain extraordinarily quiescent in the sense that they are not actively proliferating, but they fulfill the necessary role to control the permeability of their monolayer that lines the interior of blood vessels. The cell–cell junctions between ECs in the endothelium comprise tight junctions and adherens homotypic junctions, which are ubiquitous along the vascular tree. Adherens junctions are adhesive intercellular contacts that are crucial for the organization of the EC monolayer and its maintenance and regulation of normal microvascular function. The molecular components and underlying signaling pathways that control the association of adherens junctions have been described in the last few years. In contrast, the role that dysfunction of these adherens junctions has in contributing to human vascular disease remains an important open issue. Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid mediator found at high concentrations in blood which has important roles in the control of the vascular permeability, cell recruitment, and clotting that follow inflammatory processes. This role of S1P is achieved through a signaling pathway mediated through a family of G protein-coupled receptors designated as S1PR1. This review highlights novel evidence for a direct linkage between S1PR1 signaling and the mediation of EC cohesive properties that are controlled by VE-cadherin. [ABSTRACT FROM AUTHOR]

Archidendron clypearia (A. clypearia), a Fabaceae family member, is widely used as an anti-inflammatory herbal medicine; however, its antibacterial and antidiabetic properties have not been extensively investigated. This study aimed to systematically analyze the antibacterial and antidiabetic components of A. clypearia by utilizing a combination of analytical methods. First, ten different polarity extracts were analyzed through ultra-performance liquid chromatography (UPLC), and their antibacterial and antidiabetic activities were evaluated. Then the spectrum–effect relationship between the biological activity and UPLC chromatograms was analyzed by partial least squares regression and gray relational analysis, followed by corresponding validation using isolated components. Finally, network pharmacology and molecular docking were implemented to predict the main antibacterial target components of A. clypearia and the enzyme inhibition active sites of α-amylase and α-glucosidase. P15, P16, and P20 were found to be the antibacterial and antidiabetic active components. The inhibitory effect of 7-O-galloyltricetiflavan (P15) on six bacterial species may be mediated through the lipid and atherosclerosis pathway, prostate cancer, adherens junctions, and targets such as SRC, MAPK1, and AKT1. The molecular docking results revealed that 7-O-galloyltricetiflavan and 7,4′-di-O-galloyltricetiflavan (P16/P20) can bind to α-amylase and α-glucosidase pockets with binding energies lower than −6 kcal/mol. Our study provides guidance for the development of antibacterial and antidiabetic products based on A. clypearia and can be used as a reference for the evaluation of bioactivity of other herbs. [ABSTRACT FROM AUTHOR]

In recent years, the development of functional foods targeting gastrointestinal disorders has been in progress. Partially hydrolyzed guar gum (PHGG), which is a water‐soluble dietary fiber, is known to have a constipation‐improving effect. However, many aspects of the mechanism remain unclear. In this study, we investigated the role of aquaporin‐3 (AQP3), which regulates the water content of feces in ameliorative effect of PHGG on constipation. Rats were allowed to freely consume a normal diet or a diet containing 5% PHGG for 14 days, and defecation parameters were measured. We also analyzed the expression levels of genes involved in water transport in the colon. The defecation frequency and volume of rats treated with PHGG were not different from those from the control group, but the fecal water content was significantly increased, and soft stools were observed. The expressions of claudin‐1, tight junction protein‐1, and cadherin‐1, which are involved in tight junctions or adherens junctions, were almost the same in the PHGG‐treated group and the control group. The expression level of AQP3 in the colon was significantly decreased in the PHGG‐treated group. In this study, PHGG decreased the colonic AQP3 expression, thereby suppressing water transport from the luminal side to the vascular side and improving constipation. [ABSTRACT FROM AUTHOR]

In the cochlea, connexin 26 (Cx26) and connexin 30 (Cx30) co‐assemble into two types of homomeric and heteromeric gap junctions between adjacent non‐sensory epithelial cells. These channels provide a mechanical coupling between connected cells, and their activity is critical to maintain cochlear homeostasis. Many of the mutations in GJB2 or GJB6, which encode Cx26 and Cx30 in humans, impair the formation of membrane channels and cause autosomal syndromic and non‐syndromic hearing loss. Thus, deciphering the connexin trafficking pathways in situ should represent a major step forward in understanding the pathogenic significance of many of these mutations. A growing body of evidence now suggests that Cx26/Cx30 heteromeric and Cx30 homomeric channels display distinct assembly mechanisms. Here, we review the most recent advances that have been made toward unraveling the biogenesis and stability of these gap junctions in the cochlea. Key Findings: Cx26/Cx30 heteromeric and Cx30 homomeric channels display distinct assembly mechanismsCadherin‐based tricellular adherens junctions promote the microtubule‐mediated trafficking of Cx26/Cx30 oligomers to the cell surface and further assembly into GJPsEphrin‐B2 promotes the assembly and sequestration of Cx30 GJPs into caveolae membrane domains in a cell‐autonomous manner [ABSTRACT FROM AUTHOR]

Introduction: Chronic stress is co-morbid with alcohol use disorder that feedback on one another, thus impeding recovery from both disorders. Stress and the stress hormone corticosterone aggravate alcohol-induced intestinal permeability and liver damage. However, the mechanisms involved in compounding tissue injury by stress/corticosterone and alcohol are poorly defined. Here we explored the involvement of the TRPV6 channel in stress (or corticosterone) 3and alcohol-induced intestinal epithelial permeability, microbiota dysbiosis, and systemic inflammation. Methods: Chronic alcohol feeding was performed on adult wild-type and Trpv6-/- mice with or without corticosterone treatment or chronic restraint stress (CRS). The barrier function was determined by evaluating inulin permeability in vivo and assessing tight junction (TJ) and adherens junction (AJ) integrity by immunofluorescence microscopy. The gut microbiota composition was evaluated by 16S rRNA sequencing and metagenomic analyses. Systemic responses were assessed by evaluating endotoxemia, systemic inflammation, and liver damage. Results: Corticosterone and CRS disrupted TJ and AJ, increased intestinal mucosal permeability, and caused endotoxemia, systemic inflammation, and liver damage in wild-type but not Trpv6-/- mice. Corticosterone and CRS synergistically potentiated the alcohol-induced breakdown of intestinal epithelial junctions, mucosal barrier impairment, endotoxemia, systemic inflammation, and liver damage in wild-type but not Trpv6-/- mice. TRPV6 deficiency also blocked the effects of CRS and CRSmediated potentiation of alcohol-induced dysbiosis of gut microbiota. Conclusions: These findings indicate an essential role of TRPV6 in stress, corticosterone, and alcohol-induced intestinal permeability, microbiota dysbiosis, endotoxemia, systemic inflammation, and liver injury. This study identifies TRPV6 as a potential therapeutic target for developing treatment strategies for stress and alcohol-associated comorbidity. [ABSTRACT FROM AUTHOR]

Dermis fibroblasts are very sensitive to penetrating UVA radiation and induce photo-damage. To protect skin cells against this environmental damage, there is an urgent need for effective compounds, specifically targeting UVA-induced mitochondrial injury. This study aimed to analyze the effect of carnosine on the proteome of UVA-irradiated human skin fibroblast, cultured in a three-dimensional (3D) biological system recapitulating dermal compartment as a test system to investigate the altered cellular pathways after 48 h and 7 days of culture with or without carnosine treatment. The obtained results indicate that UVA dysregulates Oxidative Phosphorylation, the Fibrosis Signaling Pathway, Glycolysis I and Nrf2-mediated Oxidative Stress Response. Carnosine exercises provide a protective function against the harmful effects of UVA radiation by activating the Nrf2 pathway with the upregulations of some ROS-detoxifying enzymes such as the glutathione S-transferase (GST) protein family. Additionally, carnosine regulates the activation of the Epithelial Adherens Junction and Wound Healing Signaling Pathway by mediating the activation of structural proteins such as vinculin and zyxin as well as fibronectin 1 and collagen type XVIII alpha 1 chain against UVA-induced changes. [ABSTRACT FROM AUTHOR]

Drosophila trachea is a premier model to study tube morphogenesis. After the formation of continuous tubes, tube maturation follows. Tracheal tube maturation starts with an apical secretion pulse that deposits extracellular matrix components to form a chitin-based apical luminal matrix (aECM). This aECM is then cleared and followed by the maturation of taenidial folds. Finally, air fills the tubes. Meanwhile, the cellular junctions are maintained to ensure tube integrity. Previous research has identified several key components (ER, Golgi, several endosomes) of protein trafficking pathways that regulate the secretion and clearance of aECM, and the maintenance of cellular junctions. The Osiris (Osi) gene family is located at the Triplo-lethal (Tpl) locus on chromosome 3R 83D4-E3 and exhibits dosage sensitivity. Here, we show that three Osi genes (Osi9, Osi15, Osi19), function redundantly to regulate adherens junction (AJ) maintenance, luminal clearance, taenidial fold formation, tube morphology, and air filling during tube maturation. The localization of Osi proteins in endosomes (Rab7-containing late endosomes, Rab11-containing recycling endosomes, Lamp-containing lysosomes) and the reduction of these endosomes in Osi mutants suggest the possible role of Osi genes in tube maturation through endosome-mediated trafficking. We analyzed tube maturation in zygotic rab11 and rab7 mutants, respectively, to determine whether endosome-mediated trafficking is required. Interestingly, similar tube maturation defects were observed in rab11 but not in rab7 mutants, suggesting the involvement of Rab11-mediated trafficking, but not Rab7-mediated trafficking, in this process. To investigate whether Osi genes regulate tube maturation primarily through the maintenance of Rab11-containing endosomes, we overexpressed rab11 in Osi mutant trachea. Surprisingly, no obvious rescue was observed. Thus, increasing endosome numbers is not sufficient to rescue tube maturation defects in Osi mutants. These results suggest that Osi genes regulate other aspects of endosome-mediated trafficking, or regulate an unknown mechanism that converges or acts in parallel with Rab11-mediated trafficking during tube maturation. Author summary: The Drosophila trachea is the ideal model system to study conserved and fundamental mechanisms of tubular organ formation. In humans, tubular organs include, but are not limited to, the lungs, kidneys, and blood vessels. Defects in tube formation cause severe human diseases, such as polycystic kidney disease. After tubes begin to form, they will continue to mature to their functional lengths and diameters, and in the case of tracheae and lungs, will fill with air. The protein trafficking pathway, which is involved in delivering proteins to their final destinations, is heavily involved in various steps of the tube formation process. Previous studies have identified several key components within the protein trafficking pathway (ER, Golgi, endosomes) that regulate various processes during tube maturation. We identified the Osiris proteins, which are required for tube maturation including the maintenance of cellular junctions, clearance of the apical lumen, formation of taenidial folds to prevent the collapse of the tube, and air filling. In addition, Osi proteins regulate tube maturation potentially through endosome-mediated trafficking. This work will improve our understanding of the function of Osi genes and vesicular trafficking in the tube maturation process. [ABSTRACT FROM AUTHOR]

The cardiomyocyte phenotypic switch from a proliferative to terminally differentiated state results in the loss of regenerative potential of the mammalian heart shortly after birth. Nonmuscle myosin IIB (NM IIB)-mediated actomyosin contractility regulates cardiomyocyte cytokinesis in the embryonic heart, and NM IIB levels decline after birth, suggesting a role for cellular tension in the regulation of cardiomyocyte cell cycle activity in the postnatal heart. To investigate the role of actomyosin contractility in cardiomyocyte cell cycle arrest, we conditionally activated ROCK2 kinase domain (ROCK2:ER) in the murine postnatal heart. Here, we show that α5/β1 integrin and fibronectin matrix increase in response to actomyosinmediated tension. Moreover, activation of ROCK2:ER promotes nuclear translocation of Yap, a mechanosensitive transcriptional co-activator, and enhances cardiomyocyte proliferation. Finally, we show that reduction of myocardial α5 integrin rescues the myocardial proliferation phenotype in ROCK2:ER hearts. These data demonstrate that cardiomyocytes respond to increased intracellular tension by altering their intercellular contacts in favor of cell-matrix interactions, leading to Yap nuclear translocation, thus uncovering a function for nonmuscle myosin contractility in promoting cardiomyocyte proliferation in the postnatal heart. [ABSTRACT FROM AUTHOR]

Diabetic retinopathy (DR) is characterized by morphologic and metabolic alterations in endothelial cells (ECs) and pericytes (PCs) of the blood–retinal barrier (BRB). The loss of interendothelial junctions, increased vascular permeability, microaneurysms, and finally, EC detachment are the main features of DR. In this scenario, a pivotal role is played by the extensive loss of PCs. Based on previous results, the aim of this study was to assess possible beneficial effects exerted by adipose mesenchymal stem cells (ASCs) and their pericyte-like differentiated phenotype (P-ASCs) on human retinal endothelial cells (HRECs) in high glucose conditions (25 mM glucose, HG). P-ASCs were more able to preserve BRB integrity than ASCs in terms of (a) increased transendothelial electrical resistance (TEER); (b) increased expression of adherens junction and tight junction proteins (VE-cadherin and ZO-1); (c) reduction in mRNA levels of inflammatory cytokines TNF-α, IL-1β, and MMP-9; (d) reduction in the angiogenic factor VEGF and in fibrotic TGF-β1. Moreover, P-ASCs counteracted the HG-induced activation of the pro-inflammatory phospho-ERK1/2/phospho-cPLA2/COX-2 pathway. Finally, crosstalk between HRECs and ASCs or P-ASCs based on the PDGF-B/PDGFR-β axis at the mRNA level is described herein. Thus, P-ASCs might be considered valuable candidates for therapeutic approaches aimed at countering BRB disruption in DR. [ABSTRACT FROM AUTHOR]

Classical cadherins, including vascular endothelial (VE)-cadherin, are targeted by matrix metalloproteinases (MMPs) and γ-secretase during adherens junction (AJ) disassembly, a mechanism that might have relevance for endothelial cell (EC) integrity and vascular homeostasis. Here, we show that oxidative stress triggered by H2O2 exposure induced efficient VE-cadherin proteolysis by MMPs and γ-secretase in human umbilical endothelial cells (HUVECs). The cytoplasmic domain of VE-cadherin produced by γ-secretase, VE-Cad/CTF2—a fragment that has eluded identification so far—could readily be detected after H2O2 treatment. VE-Cad/CTF2, released into the cytosol, was tightly regulated by proteasomal degradation and was sequentially produced from an ADAM10/17-generated C-terminal fragment, VE-Cad/CTF1. Interestingly, BMP9 and BMP10, two circulating ligands critically involved in vascular maintenance, significantly reduced VE-Cad/CTF2 levels during H2O2 challenge, as well as mitigated H2O2-mediated actin cytoskeleton disassembly during VE-cadherin processing. Notably, BMP9/10 pretreatments efficiently reduced apoptosis induced by H2O2, favoring endothelial cell recovery. Thus, oxidative stress is a trigger of MMP- and γ-secretase-mediated endoproteolysis of VE-cadherin and AJ disassembly from the cytoskeleton in ECs, a mechanism that is negatively controlled by the EC quiescence factors, BMP9 and BMP10. [ABSTRACT FROM AUTHOR]

Background: Disruptions of the intestinal epithelial barrier (IEB) are frequently observed in various digestive diseases, including irritable bowel syndrome (IBS) and inflammatory bowel disease (IBD). This study assessed the improvement in the IEB during the laxative activity of phlorotannin (Pt) harvested from Ecklonia cava in constipation by examining the changes in the expression of the regulatory proteins for the tight junction (TJ) and adherens junction (AJ), and inflammatory cytokines in Sprague Dawley (SD) rats with loperamide (Lm)-induced constipation after a Pt treatment. Results: The Pt treatment induced laxative activity, including the improvement of feces-related parameters, gastrointestinal transit rate, and histological structure of the mid colon in Lm-treated SD rats. In addition, significant recovery effects were detected in the histology of IEB, including the mucus layer, epithelial cells, and lamina propria in the mid colon of Lm + Pt treated SD rats. The expression levels of E-cadherin and p120-catenin for AJ and the ZO-1, occludin, and Claudin-1 genes for TJ in epithelial cells were improved remarkably after the Pt treatment, but the rate of increase was different. Furthermore, the Pt treatment increased the expression level of several inflammatory cytokines, such as TNF-α, IL-6, IL-1β, IL-13, and IL-4 in Lm + Pt treated SD rats. Conclusions: These results provide the first evidence that the laxative activity of Pt in SD rats with Lm-induced constipation phenotypes involve improvements in the IEB. [ABSTRACT FROM AUTHOR]

The intersection of protein and lipid biology is of growing importance for understanding how cells address structural challenges during adhesion and migration. While protein complexes engaged with the cytoskeleton play a vital role, support from the phospholipid membrane is crucial for directing localization and assembly of key protein complexes. During angiogenesis, dramatic cellular remodeling is necessary for endothelial cells to shift from a stable monolayer to invasive structures. However, the molecular dynamics between lipids and proteins during endothelial invasion are not defined. Here, we utilized cell culture, immunofluorescence, and lipidomic analyses to identify a novel role for the membrane binding protein Annexin A2 (ANXA2) in modulating the composition of specific membrane lipids necessary for cortical F‐actin organization and adherens junction stabilization. In the absence of ANXA2, there is disorganized cortical F‐actin, reduced junctional Arp2, excess sprout initiation, and ultimately failed sprout maturation. Furthermore, we observed reduced filipin III labeling of membrane cholesterol in cells with reduced ANXA2, suggesting there is an alteration in phospholipid membrane dynamics. Lipidomic analyses revealed that 42 lipid species were altered with loss of ANXA2, including an accumulation of phosphatidylcholine (16:0_16:0). We found that supplementation of phosphatidylcholine (16:0_16:0) in wild‐type endothelial cells mimicked the ANXA2 knock‐down phenotype, indicating that ANXA2 regulated the phospholipid membrane upstream of Arp2 recruitment and organization of cortical F‐actin. Altogether, these data indicate a novel role for ANXA2 in coordinating events at endothelial junctions needed to initiate sprouting and show that proper lipid modulation is a critical component of these events. [ABSTRACT FROM AUTHOR]

Cell adhesion and stable signaling regulation are fundamental ways of maintaining homeostasis. Among them, the Wnt/β-CATENIN signaling plays a key role in embryonic development and maintenance of body dynamic homeostasis. At the same time, the key signaling molecule β-CATENIN in the Wnt signaling can also function as a cytoskeletal linker protein to regulate tissue barriers, cell migration, and morphogenesis. Dysregulation of the balance between Wnt signaling and adherens junctions can lead to disease. How β-CATENIN maintains the independence of these two functions, or mediates the interaction and balance of these two functions, has been explored and debated for a long time. In this study, we will focus on five aspects of β-CATENIN chaperone molecules, phosphorylation of β-CATENIN and related proteins, epithelial mesenchymal transition, β-CATENIN homolog protein γ-CATENIN and disease, thus deepening the understanding of the Wnt/β-CATENIN signaling and the homeostasis between cell adhesion and further addressing related disease problems. [ABSTRACT FROM AUTHOR]

Desmosome diseases are caused by dysfunction of desmosomes, which anchor intermediate filaments (IFs) at sites of cell--cell adhesion. For many decades, the focus of attention has been on the role of actin filament-associated adherens junctions in development and disease, especially cancer. However, interference with the function of desmosomes, their molecular constituents or their attachments to IFs has now emerged as a major contributor to a variety of diseases affecting different tissues and organs including skin, heart and the digestive tract. The first Alpine desmosome disease meeting (ADDM) held in Grainau, Germany, in October 2022 brought together international researchers from the basic sciences with clinical experts from diverse fields to share and discuss their ideas and concepts on desmosome function and dysfunction in the different cell types involved in desmosome diseases. Besides the prototypic desmosomal diseases pemphigus and arrhythmogenic cardiomyopathy, the role of desmosome dysfunction in inflammatory bowel diseases and eosinophilic esophagitis was discussed. [ABSTRACT FROM AUTHOR]

Cell junctions, including anchoring, occluding and communicating junctions, play an indispensable role in tissue architecture and homeostasis. Consequently, malfunctioning of cell junctions is linked with a wide range of disorders, including in liver. The present study was set up to investigate the effects of acute and chronic disease induced by chemical compounds on hepatic cell junctions in mice. Mice were either overdosed with paracetamol or repeatedly administered carbon tetrachloride followed by sampling at 24 hours or 8 weeks, respectively. mRNA and protein expression levels of adherens, gap and tight junction components were measured in liver using reverse transcription quantitative real-time polymerase chain reaction analysis and immunoblot techniques, respectively. It was found that protein levels of the adherens junction building blocks β-catenin and γ-catenin, the gap junction components Cx26 and Cx32, and the tight junction constituent zonula occludens 2 were decreased, while mRNA levels of the adherens junction building block E-cadherin, and the tight junction constituent zonula occludens 2 and claudin 1 were upregulated following paracetamol overdosing. Repeated administration of carbon tetrachloride increased protein levels of E-cadherin, β-catenin, Cx26, Cx32, Cx43 and claudin 1. The latter was reflected at the mRNA level. In conclusion, acute and chronic liver disease have different effects on cell junctions in liver. [ABSTRACT FROM AUTHOR]

Whole exome sequencing of invasive mammary carcinomas revealed the association of mutations in PTEN and ZFHX3 tumor suppressor genes (TSGs). We generated single and combined PTEN and ZFHX3 knock-outs (KOs) in the immortalized mammary epithelial cell line MCF10A to study the role of these genes and their potential synergy in migration regulation. Inactivation of PTEN, but not ZFHX3, induced the formation of large colonies in soft agar. ZFHX3 inactivation in PTEN KO, however, increased colony numbers and normalized their size. Cell migration was affected in different ways upon PTEN and ZFHX3 KO. Inactivation of PTEN enhanced coordinated cell motility and thus, the collective migration of epithelial islets and wound healing. In contrast, ZFHX3 knockout resulted in the acquisition of uncoordinated cell movement associated with the appearance of immature adhesive junctions (AJs) and the increased expression of the mesenchymal marker vimentin. Inactivation of the two TSGs thus induces different stages of partial epithelial-to-mesenchymal transitions (EMT). Upon double KO (DKO), cells displayed still another motile state, characterized by a decreased coordination in collective migration and high levels of vimentin but a restoration of mature linear AJs. This study illustrates the plasticity of migration modes of mammary cells transformed by a combination of cancer-associated genes. [ABSTRACT FROM AUTHOR]

For electromechanical coupling of cardiomyocytes, intercalated discs (ICDs) are pivotal as highly specialized intercellular contact areas. ICD consists of adhesive contacts, such as desmosomes and adherens junctions (AJs) that are partially intermingled and thereby form an area composita to provide mechanical strength, as well as gap junctions (GJ) and sodium channels for excitation propagation. In contrast, in epithelia, mixed junctions with features of desmosomes and AJs are regarded as transitory primarily during the formation of desmosomes. The anatomy of desmosomes is defined by a typical ultrastructure with dense intracellular plaques anchoring the cadherin‐type adhesion molecules to the intermediate filament cytoskeleton. Desmosomal diseases characterized by impaired adhesive and signalling functions of desmosomal contacts lead to arrhythmogenic cardiomyopathy when affecting cardiomyocytes and cause pemphigus when manifesting in keratinocytes or present as cardiocutaneous syndromes when both cell types are targeted by the disease, which underscores the high biomedical relevance of these cell contacts. Therefore, comparative analyses regarding the structure and regulation of desmosomal contacts in cardiomyocytes and epithelial cells are helpful to better understand disease pathogenesis. In this brief review, we describe the structural properties of ICD compared to epithelial desmosomes and suggest that mechanisms regulating adhesion may at least in part be comparable. Also, we discuss whether phenomena such as hyperadhesion or the bidirectional regulation of desmosomes to serve as signalling hubs in epithelial cells may also be relevant for ICD. [ABSTRACT FROM AUTHOR]

Mechanical stiffness is recognized as a key physical factor and directs cell function via a mechanotransduction process, from extracellular physical cues to intracellular signaling cascades that affect transcriptional activity. Cells continually receive mechanical signals from both the surrounding matrix and adjacent cells. However, how mechanical stiffness cue at cell–substrate interfaces coordinates cell–cell junctions in guiding mesenchymal stem cell behaviors is poorly understood. Here, polydimethylsiloxane substrates with different stiffnesses were used to study mechanosensation/transduction mechanisms in controlling odontogenic differentiation of dental papilla cells (DPCs). DPC phenotypes (morphology and differentiation) changed in response to the applied force derived from stiff substrates. Significantly, higher expression of paxillin on stiffer substrates promoted DPC dentinogenesis. Upon treatment with siRNA to knockdown paxillin, N-cadherin increased mainly in the cytomembrane at the area of cell–cell contacts, whereas β-catenin decreased in the nuclei. The result of a double luciferase reporter assay showed that stiffness promoted β-catenin binding to TCF, which could coactivate the target genes associated with odontogenic differentiation, as evidenced by bioinformatics analysis. Finally, we determined that the addition of a β-catenin inhibitor suppressed DPC mineralization in all the stiffness groups. Thus, our results indicated that a mechanotransduction process from cell–substrate interactions to cell–cell adhesions was required for DPC odontogenic differentiation under the stimulation of substrate stiffness. This finding suggests that stem cell fate specification under the stimulus of stiffness at the substrates is based on crosstalk between substrate interactions and adherens junctions, which provides an essential mechanism for cell-based tissue engineering. [ABSTRACT FROM AUTHOR]

Vanadium (V) exposure is known to induce renal toxicity, yet its specific effects on renal cell types and molecular mechanisms remain incompletely understood. We used single nucleus RNA sequencing (snRNA-seq) to characterize the impact of V on duck kidney cells at a cellular resolution. Following a 44-day exposure, immunofluorescence analysis revealed a significant increase in α-SMC expression in the renal interstitium, indicative of fibrotic response. SnRNA-seq identified 12 major cell types organized into 19 clusters within the kidney. Significant changes in cell composition were observed, notably an increase in proximal tubule cells (PT2 subtype), glomerular endothelial cells, principal cells, and alterations in immune cell proportions, while collecting duct intercalated cells (CD-IC) and thick ascending limb showed decreased percentages. Differential gene expression analysis highlighted pathways implicated in V toxicity across different cell types. Changes in drug metabolism-cytochrome P450, butanoate metabolism, and actin cytoskeleton regulation were exhibited by PT cells. Alterations in collecting duct secretion, oxidative phosphorylation, and bicarbonate reclamation pathways were shown in CD-IC cells. Furthermore, immune cells displayed changes in T cell receptor and chemokine signaling pathways, indicative of altered immune responses. Taken together, these findings contribute to a better shedding light on the pathogenic mechanisms of V induced renal injury. [Display omitted] • Vanadium exposure significantly alters the composition of duck kidney cells. • Vanadium exposure caused proximal tubule cells to exhibit changes in butanoate metabolism. • CD-IC cells show alterations in collecting duct secretion and oxidative phosphorylation after vanadium exposure. • Vanadium exposure alters T cell receptor and chemokine signaling pathways. [ABSTRACT FROM AUTHOR]

Aneurysm treatments are crucial to minimize the rupture risk. The underlying molecular processes mediating cellular remodeling, endothelialization, and aneurysm healing following endovascular treatment are poorly understood. The current study aims to explore circulating miRNA as a treatment and outcome-associated biomarkers in patients undergoing endovascular treatment.Patients undergoing endovascular interventions for unruptured intracranial aneurysms, using either flow diverter placement or coil embolization, were enrolled. Blood samples were collected before the intervention and during a follow-up period between 6 and 18 months. Total mRNA/miRNA was isolated from plasma, followed by RNA-seq analysis. Gene Ontology analysis was used to identify pathways linked to altered miRNA expression.Twenty-three patients participated, with 13 (56.5%) undergoing flow diversion and 10 (43.5%) coil embolization. The median follow-up sample collection time was 10.70 months (SEM ± 1.32). No significant differences in angiographic occlusion were noted between intervention groups. Differentially expressed miRNAs were not identified between groups at baseline. However, at follow-up, 39 miRNAs were upregulated and 41 were downregulated, independent of intervention. Notably, three miRNAs (miR-4746-5p, miR-4685-3p, and miR-490-3p) were downregulated in the flow diversion group compared to the coil embolization group. Bioinformatics analysis revealed associations with upregulated fluid shear stress, p53, adherens junction pathways, along with downregulated apoptosis pathways.This study suggests that fluid shear stress and apoptosis may influence aneurysm healing or thromboembolic events in flow diverter-treated patients. Further research is warranted to elucidate the functional significance of these findings in treatment outcomes, providing valuable insights for improved patient care in intracranial aneurysm management. [ABSTRACT FROM AUTHOR]