Adherens Junction Proteins Research Articles

Rac1/Wave2/Arp3 Pathway Mediates Rat Blood-Brain Barrier Dysfunction under Simulated Microgravity Based on Proteomics Strategy.

The blood-brain barrier (BBB) is critical to maintaining central nervous system (CNS) homeostasis. However, the effects of microgravity (MG) on the BBB remain unclear. This study aimed to investigate the influence of simulated MG (SMG) on the BBB and explore its potential mechanism using a proteomic approach. Rats were tail-suspended to simulate MG for 21 days. SMG could disrupt the BBB, including increased oxidative stress levels, proinflammatory cytokine levels, and permeability, damaged BBB ultrastructure, and downregulated tight junctions (TJs) and adherens junctions (AJs) protein expression in the rat brain. A total of 554 differentially expressed proteins (DEPs) induced by SMG were determined based on the label-free quantitative proteomic strategy. The bioinformatics analysis suggested that DEPs were mainly enriched in regulating the cell–cell junction and cell–extracellular matrix biological pathways. The inhibited Ras-related C3 botulinum toxin substrate 1 (Rac1)/Wiskott–Aldrich syndrome protein family verprolin-homologous protein 2 (Wave2)/actin-related protein 3 (Arp3) pathway and the decreased ratio of filamentous actin (F-actin) to globular actin contributed to BBB dysfunction induced by SMG. In the human brain microvascular endothelial cell (HBMECs), SMG increased the oxidative stress levels and proinflammatory cytokine levels, promoted apoptosis, and arrested the cell cycle phase. Expression of TJs and AJs proteins were downregulated and the distribution of F-actin was altered in SMG-treated HBMECs. The key role of the Rac1/Wave2/Arp3 pathway in BBB dysfunction was confirmed in HBMECs with a specific Rac1 agonist. This study demonstrated that SMG induced BBB dysfunction and revealed that Rac1/Wave2/Arp3 could be a potential signaling pathway responsible for BBB disruption under SMG. These results might shed a novel light on maintaining astronaut CNS homeostasis during space travel.

The Arp2/3 Inhibitory Protein Arpin Is Required for Intestinal Epithelial Barrier Integrity.

The intestinal epithelial barrier (IEB) depends on stable interepithelial protein complexes such as tight junctions (TJ), adherens junctions (AJ), and the actin cytoskeleton. During inflammation, the IEB is compromised due to TJ protein internalization and actin remodeling. An important actin regulator is the actin-related protein 2/3 (Arp2/3) complex, which induces actin branching. Activation of Arp2/3 by nucleation-promoting factors is required for the formation of epithelial monolayers, but little is known about the relevance of Arp2/3 inhibition and endogenous Arp2/3 inhibitory proteins for IEB regulation. We found that the recently identified Arp2/3 inhibitory protein arpin was strongly expressed in intestinal epithelial cells. Arpin expression decreased in response to tumor necrosis factor (TNF)α and interferon (IFN)γ treatment, whereas the expression of gadkin and protein interacting with protein C-kinase α-subunit 1 (PICK1), other Arp2/3 inhibitors, remained unchanged. Of note, arpin coprecipitated with the TJ proteins occludin and claudin-1 and the AJ protein E-cadherin. Arpin depletion altered the architecture of both AJ and TJ, increased actin filament content and actomyosin contractility, and significantly increased epithelial permeability, demonstrating that arpin is indeed required for maintaining IEB integrity. During experimental colitis in mice, arpin expression was also decreased. Analyzing colon tissues from ulcerative colitis patients by Western blot, we found different arpin levels with overall no significant changes. However, in acutely inflamed areas, arpin was significantly reduced compared to non-inflamed areas. Importantly, patients receiving mesalazine had significantly higher arpin levels than untreated patients. As arpin depletion (theoretically meaning more active Arp2/3) increased permeability, we wanted to know whether Arp2/3 inhibition would show the opposite. Indeed, the specific Arp2/3 inhibitor CK666 ameliorated TNFα/IFNγ-induced permeability in established Caco-2 monolayers by preventing TJ disruption. CK666 treatment also attenuated colitis development, colon tissue damage, TJ disruption, and permeability in dextran sulphate sodium (DSS)-treated mice. Our results demonstrate that loss of arpin triggers IEB dysfunction during inflammation and that low arpin levels can be considered a novel hallmark of acute inflammation.

Development of a novel biomarker for the early detection of colorectal cancer

Colorectal cancer (CRC) is one of the most common cancer and a leading cause of cancer related deaths both in men and in women worldwide including US. A positive family history is strongly correlated with an increased risk of CRC diagnosis during lifetime. However, CRC is an indolent disease in its early stages and usually becomes symptomatic when it progresses to more advanced stages. Conventional screening methods have several limitations and currently, there is no screening procedure available to detect CRC at a very early stage. Under normal physiological condition, the epithelial lining of the gut gets breached by various factors including food, that creates local wound. The surviving epithelial cells around the wound lose their differentiated structure and undergoes epithelial mesenchymal transition (EMT) and migrate and proliferate to cover the injured area. Once the wound is covered, the cells start differentiating back into their normal cell type and become a part of the epithelium. If the cells fail to stop proliferating following wound closure by overriding the switch to differentiating that can lead to colonic polyp, or tumor, and if not detected in a timely manner than, it leads ultimately to CRC development. Previously, we reported that β-catenin; an adherence junction protein gets phosphorylated by Janus kinase 3 (Jak3), a non-receptor tyrosine kinase, where Jak3 directly phosphorylates three tyrosine residues, viz. Tyr30, Tyr64, and Tyr86 in the N-terminal domain (NTD) of β-catenin. Now, our data show that human biopsies from colonic polyps not only associate with EMT, but also with only those mutation in β-catenin where Jak3, fail to phosphorylate β-catenin thereby making the cells override the switch and undergo uncontrolled proliferation and polyp formation. Based on this mechanistic understanding, the present study demonstrates the development of probe that detects the transition of colonic cells from proliferative stage to a differentiated (non-proliferative) stage that can diagnose CRC at the pre-polyp stage. The probe can not only play an important role in the early detection and/or treatment of patients with colorectal cancer but also increase life expectancy post detection. For this study, we collectedde-identified and discarded specimen from normal and/or polyps (n=40 cases; 20 normal and 20 polyps) through our collaboration with clinicians from local hospitals. Briefly, snap-frozen tissues were used to isolate DNA with the QIAamp DNA mini kit (Qiagen) as reported before by our group. All DNA samples were quantified using UV spectrophotometer and analyzed through agarose gel electrophoresis. The DNA sample was used as template in PCR reactions using the β-catenin-probes to amplify the regions encompassing Y30, Y64, and Y86 followed by sequencing of PCR samples. This was followed by histological analysis of colonic polyp sections from each patient stained with H&E and images were taken with phase contrast microscope. Our results show strongly correlation between mutations and histological analysis indicating the aforementioned detection of intactness of EMT switch as potential novel biomarker for the early detection of colorectal cancer.

KRIT1 stabilizes endothelial adherens junctions independent of Rap1 via regulation of β1‐integrin

The junctional scaffold KRIT1 interacts with adherens junctions (AJ) proteins to stabilize endothelial cell-cell contacts. Loss of KRIT1 function leads to Cerebral Cavernous Malformations (CCM), a disease characterized by leaky vascular lesions as the result of perturbed endothelial proliferation and inflammatory signaling. As a true Rap1 effector, KRIT1 localizes to and associates with AJ after binding active Rap1. Inhibition of this interaction by mutation of the Rap1-binding site (KRIT1-R452E) prevents KRIT1 from localizing to AJ and stabilizing barrier function. However, it remains unclear whether KRIT1 can stabilize endothelial junctions independently of Rap1. In order to study the mechanisms of KRIT1-mediated junction stabilization, we targeted mCherry-tagged KRIT1-R452E to the plasma membrane using a C-terminal lipid modification (MT-R452E). Immunofluorescence studies indicated that this construct localized to cell-cell contacts similar to WT-KRIT1 in endothelial cells depleted of endogenous KRIT1 (KRIT1 shRNA). While MT-R452E partially rescued AJ staining in these cells, it was unable to rescue barrier function, suggesting that both KRIT1 and Rap1 may be necessary at AJ to stabilize the endothelial barrier. As Rap1 binding may also regulate a putative intramolecular interaction between KRIT1's first NPxY motif and its C-terminal PTB domain, we sought to evaluate whether stabilization of endothelial junctions was dependent on KRIT1 conformation. The NPxY motif of MT-R452E was mutated (N192A-Y195A) to yield a constitutively open, membrane-targeted construct (MT-R452E-ΔNPxY). Immunofluorescence confirmed that this construct localized to cell-cell contacts, and appeared to rescue AJ staining after loss of endogenous KRIT1. Interestingly, open conformation R452E lacking any targeting sequence (R452E-ΔNPxY) also rescued AJ staining despite remaining primarily cytosolic. Further, both open conformation constructs rescued barrier function independent of Rap1 binding, indicating that KRIT1's junction stabilizing effects may be exerted without binding of Rap1 or localization to cell-cell contacts. As this NPxY (192-195) motif mediates binding to ICAP1a, which has been shown to regulate β1-integrin activity, we next examined whether junctional stability was associated with changes in β1-integrin activation. Using an activation-specific antibody against β1-integrin, we observed an increase in large fibrillar adhesions in cells depleted of KRIT1 (KRIT1 shRNA), or rescued with either R452E or MT-R452E, constructs which notably failed to rescue loss of barrier function. Conversely, expression of WT, MT-R452E-ΔNPXY, and R452E-ΔNPXY constructs, which do rescue barrier function, was associated with smaller, peripheral staining of focal contacts. These results suggest that KRIT1 may stabilize endothelial cell junctions independent of Rap1 by regulating β1-integrin-containing adhesive complexes. We expect that these novel findings will further our understanding of junctional physiology while facilitating the development of novel CCM therapeutics.

SARS‐CoV‐2 spike protein induces degradation of junctional proteins that maintain endothelial barrier integrity

BackgroundSARS‐CoV‐2 enters cells through the Angiotensin‐converting enzyme 2 (ACE‐2) receptor present on the cell surface. ACE‐2 is present in many cell types including endothelial cells, where it functions to protect against oxidative damage. Reports suggest that COVID‐19 patients also showed symptoms related to endothelial damage. We hypothesized that these vascular symptoms might be associated with disrupted endothelial barrier integrity. We investigated this in vitro using endothelial cell culture and recombinant SARS‐CoV‐2 spike protein S1 Receptor‐Binding Domain (S1RBD).Materials and MethodsMouse brain microvascular endothelial cells from normal (C57BL/6 mice) and diabetic (db/db) mice were used. The expression of VE‐Cadherin, PECAM‐1, JAM‐A and Connexin 43, was probed by Western blot in normal and diabetic cells, before and after treatment with S1RBD.ResultsThe expression of VE‐Cadherin, an endothelial adherens junction protein, was not affected by diabetes or by S1RBD treatment. PECAM‐1 expression showed a small but significant (~15%) decrease after S1RD treatment in control cells. Junctional Adhesion Molecule (JAM‐A), a tight junction protein, and the gap junction protein, Connexin‐43, decreased by ~50 and 80% respectively in the presence of S1RBD when compared to untreated controls. Expression of PECAM‐1, JAM‐A and connexin‐43 were significantly lower in untreated diabetic endothelial cells, compared to untreated control cells. This expression level further decreased significantly in the presence of S1RBD, with maximal effects observed with JAM‐A and Connexin‐43 expression (~80 and 90% decrease, respectively). These results indicate that healthy and diabetic endothelial cells respond differently when challenged with SARS‐CoV‐2 S1RBD.ConclusionS1RBD‐induces degradation of endothelial junctional proteins that likely affects endothelial barrier function and causes vascular damage.

Nanoarchitecture and Molecular Interactions of Epithelial Cell Junction Proteins Revealed by Super‐Resolution Microscopy

Epithelial cells are polarized with defined apical tight junctions (TJs), lateral adherens junctions (AJs), and basal integrin-matrix interactions. However, it is increasingly recognized that their characteristic resident proteins can be found in each other's territories and demonstrate previously unrecognized functions. This study presents the nanoarchitecture and nano-colocalization of cell junction proteins in culture and in intestinal tissue by Stochastic Optical Reconstruction Microscopy (STORM). STORM allows for resolution of approximately 20 nm in the xy plane and 50 nm in the z plane. The Z-axial view of non-cancerous MDCK-II cell AJ proteins, β-catenin and p120ctn, showed greatest expression density towards the apical milieu of AJ. This is accompanied by the distinctive displacement of p120ctn being apical to β-catenin. Towards the basal direction, p120ctn and β-catenin become increasingly colocalized. Interestingly, this topography is lost in metastatic prostate cancer PC3 cells, in that p120ctn localization becomes basal to β-catenin. The deletion of TJ protein claudin-7 is sufficient in altering the polarity of p120ctn to basal β-catenin localization, like that seen in PC3 cells compared to MDCK-II. Loss of claudin-7 also significantly altered localization and distribution of many cell junction proteins. Therefore, STORM revealed the regional nanoarchitecture of cellular junctions that were previously unavailable, providing new insights in their potential trans-compartmental modulation of protein functions.

Anti-VEGF treatment suppresses remodeling factors and restores epithelial barrier function through the E-cadherin/β-catenin signaling axis in experimental asthma models.

Besides maintaining a physical barrier with adherens junctional (AJ) and tight junctional proteins, airway epithelial cells have important roles in modulating the inflammatory processes of allergic asthma. E-cadherin and β-catenin are the key AJ proteins that are involved in airway remodeling. Various mediators such as transforming growth factor-β (TGF-β), epidermal growth factor (EGF), fibroblast growth factor (FGF), platelet derived growth factor (PDGF), insulin-like growth factor (IGF), tumor necrosis factor-α (TNF-α) and angiogenic factors, such as vascular endothelial growth factor (VEGF), are released by the airway epithelium in allergic asthma. The signaling pathways activated by these growth factors trigger epithelial-mesenchymal transition (EMT), which contributes to fibrosis and subsequent downregulation of E-cadherin. The present study used a mouse asthma model to investigate the effects of anti-VEGF, anti-TNF and corticosteroid therapies on growth factor and E-cadherin/β-catenin expression. The study used 38 male BALB/c mice, divided into 5 groups. A chronic mouse asthma model was created by treating 4 of the groups with inhaled and intraperitoneal ovalbumin (n= 8 per group). Saline, anti-TNF-α (etanercept), anti-VEGF (bevacizumab) or a corticosteroid (dexamethasone) were applied to each group by intraperitoneal injection. No medication was administered to the control group (n=6). Immunohistochemistry for E-cadherin, β-catenin and growth factors was performed on lung tissues and protein expression levels assessed using H-scores. Statistically significant differences were observed in E-cadherin, β-catenin, EGF, FG, and PFGF (P<0.001 for all) as well as the IGF H-scores between the five groups (P<0.005). Only anti-VEGF treatment caused E-cadherin and β-catenin levels to increase to the level of non-asthmatic control groups (P>0.005). All treatment groups had reduced TGF-β, PDGF and FGF H-scores in comparison with the untreated asthma group (P=0.001). The EGF and IGF levels were not significantly different between the untreated asthmatic and non-asthmatic controls. The results suggested that anti-VEGF and TNF-α inhibition treatments are effective in decreasing growth factors, in a similar manner to conventional corticosteroid treatments. Anti-VEGF and TNF inhibition therapy may be an effective treatment for remodeling in asthma while offering an alternative therapeutic option to steroid protective agents. The data suggested that anti-VEGF treatment offered greater restoration of the epithelial barrier than both anti-TNF-α and corticosteroid treatment.

Ethanol-induced lymphatic endothelial cell permeability via MAP-kinase regulation.

Chronic alcohol alters the immune system enhancing the susceptibility to inflammation, bacterial, and viral infections in alcohol users. We have shown that alcohol causes increased permeability of mesenteric lymphatic vessels in alcohol-fed rats. The mechanisms of alcohol-induced lymphatic leakage are unknown. Endothelial cell monolayer permeability is controlled by junctional proteins complexes called tight junctions (TJ) and adherens junctions (AJ), and each can be regulated by MAPK activation. We hypothesize that ethanol induces lymphatic endothelial cell (LEC) permeability via disruption of LEC TJ through MAPK activation. An in vitro model of rat LECs was used. Ethanol-supplemented medium was added at concentrations of 0, 25, and 50 mM to confluent cells. Resistance-based barrier function, transwell permeability, cell viability, TJ, AJ, and MAPK protein activity, TJ and AJ gene expressions, and the role of p38 MAPK in barrier function regulation were measured. Ethanol increased the permeability of LECs compared to controls that was not associated with decreased cell viability. LECs treated with 50 mM ethanol showed an increase in phosphorylated levels of p38. No significant changes in TJ and AJ gene or protein expressions were observed after ethanol treatment. p38 inhibition prevented ethanol-induced increases in permeability. These findings suggest that p38 may play a role in the regulation of ethanol-induced LEC permeability, but altered permeability may not be associated with decreased TJ or AJ protein expression. Further investigation into junctional protein localization is needed to better understand the effects of ethanol on lymphatic endothelial cell-to-cell contacts and hyperpermeability.

Adherens junction engagement regulates functional patterning of the cardiac pacemaker cell lineage.

Cardiac pacemaker cells (CPCs) rhythmically initiate the electrical impulses that drive heart contraction. CPCs display the highest rate of spontaneous depolarization in the heart despite being subjected to inhibitory electrochemical conditions that should theoretically suppress their activity. While several models have been proposed to explain this apparent paradox, the actual molecular mechanisms that allow CPCs to overcome electrogenic barriers to their function remain poorly understood. Here, we have traced CPC development at single-cell resolution and uncovered a series of cytoarchitectural patterning events that are critical for proper pacemaking. Specifically, our data reveal that CPCs dynamically modulate adherens junction (AJ) engagement to control characteristics including surface area, volume, and gap junctional coupling. This allows CPCs to adopt a structural configuration that supports their overall excitability. Thus, our data have identified a direct role for local cellular mechanics in patterning critical morphological features that are necessary for CPC electrical activity.

Graphitic Carbon Nitride Causes Widespread Global Molecular Changes in Epithelial and Fibroblast Cells.

For scaffold and imaging applications, nanomaterials such as graphene and its derivatives have been widely used. Graphitic carbon nitride (g-C3N4) is among one such derivative of graphenes, which draws strong consideration due to its physicochemical properties and photocatalytic activity. To use g-C3N4 for biological applications, such as molecular imaging or drug delivery, it must interact with the epithelium, cross the epithelial barrier, and then come in contact with the extracellular matrix of the fibroblast cells. Thus, it becomes essential to understand its molecular mechanism of action. Hence, in this study, to understand the molecular reprogramming associated with g-C3N4, global gene expression using DNA microarrays and proteomics using tandem mass tag (TMT) labeling and mass spectrometry were performed in epithelial and fibroblast cells, respectively. Our results showed that g-C3N4 can cross the epithelial barrier by regulating the adherens junction proteins. Further, using g-C3N4–PDMS scaffolds as a mimic of the extracellular matrix for fibroblast cells, the common signaling pathways were identified between the epithelium and fibroblast cells. These pathways include Wnt signaling, integrin signaling, TGF-β signaling, cadherin signaling, oxidative stress response, ubiquitin proteasome pathway, and EGF receptor signaling pathways. These altered signature pathways identified could play a prominent role in g-C3N4-mediated cellular interactions in both epithelial and fibroblast cells. Additionally, β catenin, EGFR, and MAP2K2 protein–protein interaction networks could play a prominent role in fibroblast cell proliferation. The findings could further our knowledge on g-C3N4-mediated alterations in cellular molecular signatures, enabling the potential use of these materials for biological applications such as molecular imaging and drug delivery.

Alterations in the Gut-Microbial-Inflammasome-Brain Axis in a Mouse Model of Alzheimer's Disease.

Alzheimer’s disease (AD), a progressive neurodegenerative disorder characterized by memory loss and cognitive decline, is a major cause of death and disability among the older population. Despite decades of scientific research, the underlying etiological triggers are unknown. Recent studies suggested that gut microbiota can influence AD progression; however, potential mechanisms linking the gut microbiota with AD pathogenesis remain obscure. In the present study, we provided a potential mechanistic link between dysbiotic gut microbiota and neuroinflammation associated with AD progression. Using a mouse model of AD, we discovered that unfavorable gut microbiota are correlated with abnormally elevated expression of gut NLRP3 and lead to peripheral inflammasome activation, which in turn exacerbates AD-associated neuroinflammation. To this end, we observe significantly altered gut microbiota compositions in young and old 5xFAD mice compared to age-matched non-transgenic mice. Moreover, 5xFAD mice demonstrated compromised gut barrier function as evident from the loss of tight junction and adherens junction proteins compared to non-transgenic mice. Concurrently, we observed increased expression of NLRP3 inflammasome and IL-1β production in the 5xFAD gut. Consistent with our hypothesis, increased gut–microbial–inflammasome activation is positively correlated with enhanced astrogliosis and microglial activation, along with higher expression of NLRP3 inflammasome and IL-1β production in the brains of 5xFAD mice. These data indicate that the elevated expression of gut–microbial–inflammasome components may be an important trigger for subsequent downstream activation of inflammatory and potentially cytotoxic mediators, and gastrointestinal NLRP3 may promote NLRP3 inflammasome-mediated neuroinflammation. Thus, modulation of the gut microbiota may be a potential strategy for the treatment of AD-related neurological disorders in genetically susceptible hosts.

The role of mucosal barriers in human gut health.

The intestinal mucosa is continuously exposed to a large number of commensal or pathogenic microbiota and foreign food antigens. The intestinal epithelium forms a dynamic physicochemical barrier to maintain immune homeostasis. To efficiently absorb nutrients from food, the epithelium in the small intestine has thin, permeable layers spread over a vast surface area. Epithelial cells are renewed from the crypt toward the villi, accompanying epithelial cell death and shedding, to control bacterial colonization. Tight junction and adherens junction proteins provide epithelial cell-cell integrity. Microbial signals are recognized by epithelial cells via toll-like receptors. Environmental signals from short-chain fatty acids derived from commensal microbiota metabolites, aryl hydrocarbon receptors, and hypoxia-induced factors fortify gut barrier function. Here we summarize recent findings regarding various environmental factors for gut barrier function. Further, we discuss the role of gut barriers in the pathogenesis of human intestinal disease and the challenges of therapeutic strategies targeting gut barrier restoration.

Regulation of apical constriction via microtubule- and Rab11-dependent apical transport during tissue invagination.

The formation of an epithelial tube is a fundamental process for organogenesis. During Drosophila embryonic salivary gland (SG) invagination, Folded gastrulation (Fog)-dependent Rho-associated kinase (Rok) promotes contractile apical myosin formation to drive apical constriction. Microtubules (MTs) are also crucial for this process and are required for forming and maintaining apicomedial myosin. However, the underlying mechanism that coordinates actomyosin and MT networks still remains elusive. Here, we show that MT-dependent intracellular trafficking regulates apical constriction during SG invagination. Key components involved in protein trafficking, such as Rab11 and Nuclear fallout (Nuf), are apically enriched near the SG invagination pit in a MT-dependent manner. Disruption of the MT networks or knockdown of Rab11 impairs apicomedial myosin formation and apical constriction. We show that MTs and Rab11 are required for apical enrichment of the Fog ligand and the continuous distribution of the apical determinant protein Crumbs (Crb) and the key adherens junction protein E-Cadherin (E-Cad) along junctions. Targeted knockdown of crb or E-Cad in the SG disrupts apical myosin networks and results in apical constriction defects. Our data suggest a role of MT- and Rab11-dependent intracellular trafficking in regulating actomyosin networks and cell junctions to coordinate cell behaviors during tubular organ formation.

Impaired airway epithelial barrier integrity was mediated by PI3Kδ in a mouse model of lipopolysaccharide-induced acute lung injury

Cell-cell junctions are critical for the maintenance of cellular as well as tissue polarity and integrity. Dysfunction of airway epithelial barrier has been shown to be involved in the pathogenesis of acute lung injury (ALI). Yet the role of phosphatidylinositol 3-kinase delta (PI3Kδ) in dysregulation of airway epithelial barrier integrity in ALI has not been addressed. Mice were subjected to intratracheal instillation of lipopolysaccharide (LPS) to generate a ALI model. Two pharmacological inhibitors of PI3Kδ, IC87114 and AMG319, were respectively given to the mice. Expression of p110δ and its downstream substrate phospho-AKT (Ser473) was increased in LPS-exposed lungs. These increases were inhibited by IC87114 or AMG319. LPS led to pronounced lung injury that was accompanied by significant airway neutrophil recruitment and bronchial epithelial morphological alterations 72 h after exposure. We also found compromised expression of adherens junction protein E-cadherin and tight junction protein claudin-2 in the airway epithelial cells. Treatment with either IC87114 or AMG319 not only attenuated LPS-induced edema, lung injury and neutrophilc inflammation, reduced total protein concentration and IL-6, TNF-α secretion in BALF, but also restored epithelial E-cadherin and claudin-2 expression. In summary, our results showed that LPS can induce a delayed effect on airway epithelial barrier integrity that is mediated by PI3Kδ in a mouse model of ALI.

Effect of irradiation on the expression of E-cadherin and \u03b2-catenin in early and late radiation sequelae of the urinary bladder and its modulation by NF-\u03baB inhibitor thalidomide

PurposeIn a previous study we have shown in a mouse model that administration of nuclear factor-kappa B (NF-κB) inhibitor thalidomide has promising therapeutic effects on early radiation cystitis (ERC) and late radiation sequelae (LRS) of the urinary bladder. The aim of this study was to evaluate in the same mice the effect of thalidomide on adherens junction (AJ) proteins in ERC and LRS.MethodsUrothelial expressions of E‑cadherin and β‑catenin were assessed by immunohistochemistry in formalin-fixed paraffin-embedded (FFPE) bladder specimens over 360 days post single-dose irradiation on day 0. First, the effect of irradiation on AJ expression and then effects of thalidomide on irradiation-induced AJ alterations were assessed using three different treatment times.ResultsIrradiation provoked a biphasic upregulation of E‑cadherin and β‑catenin in the early phase. After a mild decrease of E‑cadherin and a pronounced decrease of β‑catenin at the end of the early phase, both increased again in the late phase. Early administration of thalidomide (day 1–15) resulted in a steeper rise in the first days, an extended and increased expression at the end of the early phase and a higher expression of β‑catenin alone at the beginning of the late phase.ConclusionUpregulation of AJ proteins is an attempt to compensate irradiation-induced impairment of urothelial barrier function. Early administration of thalidomide improves these compensatory mechanisms by inhibiting NF-κB signaling and its interfering effects.

Dragon's Blood Regulates Rac1-WAVE2-Arp2/3 Signaling Pathway to Protect Rat Intestinal Epithelial Barrier Dysfunction Induced by Simulated Microgravity.

Dragon’s Blood is a red resin from Dracaena cochinchinensis (Lour.) S.C. Chen (Yunnan, China). As a traditional Chinese medicinal herb, it has shown protective effects on intestinal disorders. Microgravity could alter intestinal homeostasis. However, the potential herbal drugs for preventing intestine epithelial barrier (IEB) dysfunction under microgravity are not available. This study aimed to investigate the effects of Dragon’s Blood (DB) on microgravity-induced IEB injury and explore its underlying mechanism. A rat tail-suspension model was used to simulate microgravity (SMG). Histomorphology, ultrastructure, permeability, and expression of junction proteins in jejunum, ileum, and colon of SMG rats were determined. Proteomic analysis was used to identify differentially expressed proteins (DEPs) in rat ileum mucosa altered by DB. The potential mechanism of DB to protect IEB dysfunction was validated by western blotting. The effects of several components in DB were evaluated in SMG-treated Caco-2 cells. DB protected against IEB disruption by repairing microvilli and crypts, inhibiting inflammatory factors, lowering the permeability and upregulating the expression of tight and adherens junction proteins in the ileum of SMG rats. Proteomic analysis showed that DB regulated 1080 DEPs in rat ileum mucosa. DEPs were significantly annotated in cell–cell adhesion, focal adhesion, and cytoskeleton regulation. DB increased the expression of Rac1-WAVE2-Arp2/3 pathway proteins and F-actin to G-actin ratio, which promoted the formation of focal adhesions. Loureirin C in DB showed a protective effect on epithelial barrier injury in SMG-treated Caco-2 cells. DB could protect against IEB dysfunction induced by SMG, and its mechanism is associated with the formation of focal adhesions mediated by the Rac1-WAVE2-Arp2/3 pathway, which benefits intestinal epithelial cell migration and barrier repair.

PCB and TCDD derived embryonic cardiac defects result from a novel AhR pathway

Polychlorinated biphenyls (PCBs) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) are environmental contaminants known to impact cardiac development, a key step in the embryonic development of most animals. To date, little is understood of the molecular mechanism driving the observed cardiac defects in exposed fishes. The literature shows PCB & TCDD derived cardiac defects are concurrent with, but not caused by, expression of cyp1A, due to activation of the aryl hydrocarbon receptor (AhR) gene activation pathway. However, in this study, detailed visualization of fish hearts exposed to PCBs and TCDD show that, in addition to a failure of cardiac looping in early heart development, the inner endocardial lining of the heart fails to maintain proper cell adhesion and tissue integrity. The resulting gap between the endocardium and myocardium in both zebrafish and Atlantic sturgeon suggested functional faults in endothelial adherens junction formation. Thus, we explored the molecular mechanism triggering cardiac defects using immunohistochemistry to identify the location and phosphorylation state of key regulatory and adhesion molecules. We hypothesized that PCB and TCDD activates AhR, phosphorylating Src, which then phosphorylates the endothelial adherens junction protein, VEcadherin. When phosphorylated, VEcadherin dimers, found in the endocardium and vasculature, separate, reducing tissue integrity. In zebrafish, treatment with PCB and TCDD contaminants leads to higher phosphorylation of VEcadherin in cardiac tissue suggesting that these cells have reduced connectivity. Small molecule inhibition of Src phosphorylation prevents contaminant stimulated phosphorylation of VEcadherin and rescues both cardiac function and gross morphology. Atlantic sturgeon hearts show parallels to contaminant exposed zebrafish cardiac phenotype at the tissue level. These data suggest that the mechanism for PCB and TCDD action in the heart is, in part, distinct from the canonical mechanism described in the literature and that cardiac defects are impacted by this nongenomic mechanism.

Potential targeted therapy: The role of MicroRNAs in breast cancer metastasis via epithelial-mesenchymal transition and cancer stem cell regulation

Abstract&#x0D; In the advancement of breast cancer treatment, metastatic breast cancer is remaining as an incurable disease. It contributes to almost 90% of cancer-related death in breast cancer cases. Epithelial to Mesenchymal Transition (EMT) is a serial change of the epithelial cell to gain the mesenchymal-like phenotype. In cancer, the cells that undergo the EMT lose the adherent junction protein, cell polarity, and gain the invasive phenotype. Recent studies showed that the EMT induces the cancer stem cell-like phenotypes in cancer cells. These cells possess self-renewal ability, and multi-lineage differentiation capacity to generate the new bulk of tumor during cancer distant metastasis. Both EMT and cancer stem cells take responsibility in drug-resistant, and relapse cases in breast cancer. In the last decades, a new type of non-coding RNA, microRNA (miR) shows have an important role in the normal physiological and pathophysiological condition such as cancer. Recent studies revealed that the EMT is regulated by microRNAs. In this review, we discussed the microRNAs regulation on the EMT process through TGF-β, and Wnt signaling pathways in breast cancer. Understanding of microRNA regulation in EMT in breast cancer metastasis gives a chance to explore a new therapy approach to improve the prognosis of breast cancer patients. In addition, we also explored several potential approaches targeting microRNA as a new approach of cancer treatment.&#x0D; &#x0D; Keywords: breast cancer, microRNA, EMT, metastasis, targeted therapy.

CADHERIN-11, AN ESSENTIAL REGULATOR OF CELL-CELL ADHESION UPREGULATED IN IBD PATIENTS, PLAYS ESSENTIAL ROLES IN PROMOTING FIBROBLAST ACTIVATION AND DEVELOPMENT OF INTESTINAL INFLAMMATION AND FIBROSIS

Intestinal fibrosis is a severe complication of inflammatory bowel diseases (IBD) leading to intestinal strictures and need for surgery. No effective anti-fibrotic therapy is available. Cadherin-11 (Cad-11) is an adherens junction protein, which is upregulated in rheumatoid arthritis (RA), idiopathic pulmonary fibrosis (IPF) and skin fibrosis. Inhibition of cadherin-11 has shown beneficial effects in RA and IPF animal models. A phase II clinical trial of cadherin-11 inhibition in RA has shown a good safety profile.

Diammonium glycyrrhizinate lipid ligand ameliorates lipopolysaccharide-induced acute lung injury by modulating vascular endothelial barrier function.

The aim of the present study was to investigate the effects of diammonium glycyrrhizinate lipid ligand (DGLL) treatment on acute lung injury (ALI) and pulmonary edema induced by lipopolysaccharide (LPS) in Sprague-Dawley rats. Rats orally received 30, 60 and 120 mg/kg DGLL. After 1 h, the rat ALI model was established by LPS (10 mg/kg) intraperitoneal injection. After 6 h, lung injury was evaluated using hematoxylin and eosin staining techniques. Pulmonary edema was evaluated using lung wet-dry weight ratio, protein concentrations in the bronchoalveolar lavage fluid (BALF) and Evans blue (EB) extravasation in lung tissue. The expression levels of tumor necrosis factor (TNF)-α and interleukin (IL)-1β in lung tissues were measured using ELISA. Myeloperoxidase (MPO) expression levels were detected by immunohistochemical staining. Western blotting was used to measure the expression level changes of intercellular adhesion molecule (ICAM)-1, as well as adherent and tight junction proteins, including vascular endothelial (VE)-cadherin, zonula occludens (ZO)-1, occludin and junctional adhesion molecule (JAM)-1 that were associated with pulmonary inflammation and microvascular permeability. DGLL treatment significantly alleviated ALI induced by LPS, which was demonstrated by reduction of MPO-positive cells and expression levels of TNF-α, IL-1β and ICAM-1 in rat lung tissues. In addition, DGLL abrogated LPS-induced pulmonary edema, decreased the protein concentration in BALF and reduced EB extravasation. DGLL also reversed the reduced expression of VE-cadherin and tight junction proteins, including ZO-1, occludin and JAM-1 in the lung tissues caused by LPS. In conclusion, DGLL exhibits a protective effect on LPS-induced rat ALI, which is associated with the inhibition of inflammatory cell infiltration and microvascular barrier disruption. The present results provide a theoretical basis for the application of DGLL for the potential clinical treatment of ALI.