Role of matriptase in the regulation of epithelial barrier permeability studied using MDCK cells

The!typeNII!transmembrane!serine!protease!matriptase!plays!an!important!role!in! the!integrity!of!epithelial!barriers.!However,!the!molecular!mechanisms!underlying! the!role!of!matriptase!are!unknown.! To! study! these!mechanisms,! two!variants!of!MadinNDarby!canine!kidney!(MDCK)! cells!were! used,! together!with! the! “calciumNswitch”!model! of! epithelial! function! with!measurements!of!transepithelial!electrical!resistance!(TEER).! Inhibitors!of!matriptase!proteolytic!activity!delayed!the!restoration!of!TEER!after! calciumNswitch! in!MDCKNI,! which! develop! high! TEER! and! lack! the! “leaky”! tight! junction!protein!claudinN2,!but!not!MDCKNII.!This!effect!was!confirmed!in!MDCKNI,! established!to!stably!express!matriptase!targeted!shRNA.! The!influence!of!matriptase!inhibition!on!MDCKNI!was!shown!not!to!involve!altered! expression! or! assembly! of! relevant! components! of! paracelluar! junctions! or! cytoskeleton.! This! excluded! a! role! for! claudinN2! in! the! function! of! matriptase,! which!had!previously!been!shown!in!human!CacoN2!cells,!and!this!was!confirmed! using!MDCKNI!cells!stably!overexpressing!claudinN2.! To! investigate! the! claudinN2Nindependent! function! of! matriptase,! a! candidate! substrate! approach! was! used.! Proteolytic! activation! of! proNHGF/cNMet,! PARN2,! ENaC,!EGFR!and!prostasin!by!matriptase!has!effects!on!epithelial!cell!function,!but! none!were! found! to!have!a! role! in!matriptase! restoration!of!TEER!after! calciumN switch!in!MDCKNI!cells.!As!the!direct!proteolytic!target!of!matriptase!could!not!be! identified,! potential! mediators! were! studied! using! arrays! for! phosphorylated! signalling!proteins!and!inflammatory!cytokines.!ILN1β!and!complement!component! C5!were! identified!as!genes!downregulated!by!matriptase! inhibition,!while! ILN13! showed!upregulation.! This!work!has!confirmed!the!key!role!of!matriptase!activity!in!regulating!epithelial! barrier!integrity.!The!differential!properties!of!MDCKNI!and!MDCKNII!cells!excluded! a! role! for! claudinN2.! None! of! the! known! proteolytic! targets! of! matriptase! were! involved,!however,!changes!in!cytokine!gene!expression!may!be!a!potential!route! for!matriptase!effects!on!epithelial!barrier!maintenance

Protease signaling regulates apical cell extrusion, cell contacts, and proliferation in epithelia.

Mechanisms that sense and regulate epithelial morphogenesis, integrity, and homeostasis are incompletely understood. Protease-activated receptor 2 (Par2), the Par2-activating membrane-tethered protease matriptase, and its inhibitor, hepatocyte activator inhibitor 1 (Hai1), are coexpressed in most epithelia and may make up a local signaling system that regulates epithelial behavior. We explored the role of Par2b in matriptase-dependent skin abnormalities in Hai1a-deficient zebrafish embryos. We show an unexpected role for Par2b in regulation of epithelial apical cell extrusion, roles in regulating proliferation that were opposite in distinct but adjacent epithelial monolayers, and roles in regulating cell-cell junctions, mobility, survival, and expression of genes involved in tissue remodeling and inflammation. The epidermal growth factor receptor Erbb2 and matrix metalloproteinases, the latter induced by Par2b, may contribute to some matriptase- and Par2b-dependent phenotypes and be permissive for others. Our results suggest that local protease-activated receptor signaling can coordinate cell behaviors known to contribute to epithelial morphogenesis and homeostasis

The Impact of Acute Matriptase Inhibition in Hepatic Inflammatory Models

Purpose. Dysfunction of matriptase-2 can be involved in iron regulatory disorder via downregulation of hepcidin expression. In the present study, we investigated the effects of 3-amidinophenylalanine-derived matriptase inhibitors on porcine hepatic inflammatory cell models. Methods. Hepatocyte-Kupffer cell cocultures (ratio of 2 : 1 and 6 : 1) were treated with four structurally related matriptase inhibitors at 50 μM. Cell cytotoxicity and relative expressions of IL-6 and IL-8 and the levels of hepcidin were determined by MTS and porcine-specific ELISA. The extracellular H2O2 contents were analyzed by Amplex Red method. Results. Matriptase inhibitors at 50 µM for 24 h did not increase cell death rate. The elevated ROS production observed after short-term application of inhibitor MI-441 could be correlated with lowered hepcidin expression. MI-460 could significantly enhance hepcidin levels in the supernatants of cocultures (by 62.21±26.8% in hepatocyte-Kupffer cell, 2 : 1, and by 42.6±14.3% in hepatocyte-Kupffer cell, 6 : 1, cocultures, resp.). No significant changes were found in IL-6 and IL-8 levels in cocultures exposed to matriptase inhibitors. Conclusions. Based on in vitro findings, administration of MI-460 via modulation of hepcidin expression without cytotoxic and oxidative stress inducing properties might be a reliable alternative to treat iron overload in human and veterinary clinical practice

Desmoglein 2 is less important than desmoglein 3 for keratinocyte cohesion.

Desmosomes provide intercellular adhesive strength required for integrity of epithelial and some non-epithelial tissues. Within the epidermis, the cadherin-type adhesion molecules desmoglein (Dsg) 1-4 and desmocollin (Dsc) 1-3 build the adhesive core of desmosomes. In keratinocytes, several isoforms of these proteins are co-expressed. However, the contribution of specific isoforms to overall cell cohesion is unclear. Therefore, in this study we investigated the roles of Dsg2 and Dsg3, the latter of which is known to be essential for keratinocyte adhesion based on its autoantibody-induced loss of function in the autoimmune blistering skin disease pemphigus vulgaris (PV). The pathogenic PV antibody AK23, targeting the Dsg3 adhesive domain, led to profound loss of cell cohesion in human keratinocytes as revealed by the dispase-based dissociation assays. In contrast, an antibody against Dsg2 had no effect on cell cohesion although the Dsg2 antibody was demonstrated to interfere with Dsg2 transinteraction by single molecule atomic force microscopy and was effective to reduce cell cohesion in intestinal epithelial Caco-2 cells which express Dsg2 as the only Dsg isoform. To substantiate these findings, siRNA-mediated silencing of Dsg2 or Dsg3 was performed in keratinocytes. In contrast to Dsg3-depleted cells, Dsg2 knockdown reduced cell cohesion only under conditions of increased shear. These experiments indicate that specific desmosomal cadherins contribute differently to keratinocyte cohesion and that Dsg2 compared to Dsg3 is less important in this context

Claudins in intestines

Intestines are organs that not only digest food and absorb nutrients, but also provide a defense barrier against pathogens and noxious agents ingested. Tight junctions (TJs) are the most apical component of the junctional complex, providing one form of cell-cell adhesion in enterocytes and playing a critical role in regulating paracellular barrier permeability. Alteration of TJs leads to a number of pathophysiological diseases causing malabsorption of nutrition and intestinal structure disruption, which may even contribute to systemic organ failure. Claudins are the major structural and functional components of TJs with at least 24 members in mammals. Claudins have distinct charge-selectivity, either by tightening the paracellular pathway or functioning as paracellular channels, regulating ions and small molecules passing through the paracellular pathway. In this review, we have discussed the functions of claudin family members, their distribution and localization in the intestinal tract of mammals, their alterations in intestine-related diseases and chemicals/agents that regulate the expression and localization of claudins as well as the intestinal permeability, which provide a therapeutic view for treating intestinal diseases

The involvement of the three main inflammatory bowel disease pathways and the secretion of trypsin proteolytic activity on intestinal epithelial cells

Les maladies inflammatoires chroniques de l'intestin (MICI) se caractérisent par une inflammation sévère de l'intestin grêle et du côlon et comprennent la maladie de Crohn (MC) et la rectocolite hémorragique (RCH). Les MICI sont des maladies complexes faisant intervenir des facteurs génétiques : certains senseurs bactériens, l'autophagie et le stress du réticulum endoplasmique. Un défaut de barrière de l'épithélium digestif est également fortement impliqué dans la physiopathologie du processus inflammatoire. La fonction barrière de l'épithélium digestif est assurée par plusieurs types cellulaires, synthétisant entre autres, des peptides antimicrobiens (PAM) et des mucines. Dans les MICI, une augmentation de la perméabilité intestinale et une perte de muco-sécrétion ont été décrites. Les protéases jouent un rôle fondamental dans la digestion du bol alimentaire mais également dans le maintien de l'homéostasie intestinale en activant ou dégradant divers motifs moléculaires, ou in induisant des signaux spécifiques aux cellules par l'activation de quatre récepteurs : les PARs (Protease-Activated Receptor). Dans les MICI, un excès d'activité protéolytique de type trypsine est observé. L'origine de cette activité est théoriquement attribuée aux cellules immunitaires, à une surproduction pancréatique ou au microbiote, mais les cellules épithéliales intestinales semblent également être une source majeure de protéases. L'objectif de mon projet de thèse visait à étudier l'impact des principales voies impliquées dans les MICI sur l'homéostasie des protéases épithéliales et le rôle de celles-ci dans la déstabilisation de la fonction de barrière. Nos résultats ont confirmé un excès de protéases à sérine dans les cellules épithéliales de patients atteint de MC ou de RCH. In vitro, sur des monocouches de cellules Caco-2, l'induction de l'autophagie diminuait la libération apicale de protéase de type trypsine, alors que le senseur bactériens NOD2 n'avait aucun effet. A l'inverse, une stimulation du Stress du réticulum endoplasmique (SRE) par la Thapsigargin, induisait une libération accrue de protéases actives de type trypsine au pôle apical des cellules. L'utilisation d'ABP (Activity-based probe), emprisonnant les protéases actives de type trypsine dans des surnageants apicaux de Caco-2 stimulées par la Thapsigargin, a montré une importante sécrétion d'une protéase unique au poids moléculaire de 33-KDa. Par western blot, la présence augmentée de Trypsine-3 était identifiée dans ces surnageants, de même que dans les colonocytes de patients atteints de RCH comparé à des échantillons contrôles ou CD. Seul les colonocytes de patients RCH présentaient également une induction du SRE. Sur les monocouches de Caco-2, l'induction du SRE augmentait la perméabilité paracellulaire, la sécrétion de CXCL88 et l'expression de PAM, de mucine et des récepteurs PAR2 et -4. Les inhibiteurs de protéases de type trypsine supprimaient l'augmentation de la perméabilité et l'expression des PAM, de la mucine 2 et des récepteurs PAR2 et -4 induite par le SRE, et aggravaient la sécrétion de CXCL8. Les antagonistes sélectifs des récepteurs PAR2 et/ou PAR4 inhibaient l'augmentation de la perméabilité et l'expression des PAM, de la mucine 2 et des récepteurs PAR2 et -4 induite par le SRE, mais aggravaient la sécrétion de CXCL8. Enfin, la Trypsine-3 ne modifiait pas les marqueurs de SRE. En conclusion, l'induction d'un SRE dans les cellules épithéliales déclenche une libération apicale de Trypsine-3 et d'activité trypsine, responsable de l'altération de la fonction de barrière de la monocouche cellulaire. Nous avons identifié l'implication des récepteurs PAR2 et -4 (tous deux activables par la Trypsine-3) dans la rupture de l'homéostasie de l'épithélium intestinal. La Trypsine-3 semble être spécifiquement surexprimée dans les colonocytes de patients RCH, cette surexpression pourrait être liée à une induction anormale du SRE.Crohn's disease (CD) and Ulcerative colitis (UC) are two forms of Inflammatory Bowel Disease (IBD), a chronic inflammatory pathology affecting the digestive tract. Patients suffer from relapsing flares, diarrhea, abdominal pain and bleeding. Although the molecular mechanisms of IBD are poorly understood, recent data suggest that IBD occurs in genetically predisposed individuals developing an abnormal immune response to intestinal microbes after, being exposed to specific environmental triggers. Genetic studies have reported more than 170 polymorphisms susceptible to be involved in IBD pathogenesis. The strongest associations have highlighted three main pathways altered in IBD including bacterial sensing (NOD2, CD), autophagy (ATG16L1 and IRGM, CD) and endoplasmic reticulum stress (ER-Stress) (XBP1, UC). The role of intestinal barrier function is also strongly implicated in IBD pathogenesis, and is modulated by factors present in the lumen derived from microbiota, food or at a molecular level, by factors such as proteases. In IBD pathophysiology, the inflammatory process is characterized by impaired intestinal biology including disruption of tight junctions and leaky gut, decreased amount of Paneth and Goblet cells, and translocation of luminal antigens triggering inflammation. Previous studies have demonstrated an increased level of active serine proteases in the stools and tissues of IBD patients, supposing that proteases originate from infiltrated immune cells, pancreatic secretion or microbiota. However, our team has reported that intestinal epithelial cells are a major source of serine proteases, in particular trypsin-like enzymes, are released by a stressed epithelium in pathogenic context such as irritable bowel syndrome. In this project, we aimed at better understanding whether the three main pathways involved in IBD (Nod2, autophagy, ER-stress) could be linked to an epithelial release of trypsin and reciprocally, if epithelial trypsin is able to induce or modulate these three IBD pathways. We confirmed that trypsin-like activity was significantly higher in biopsies from UC and CD patients compared to healthy controls. In Caco-2 monolayers cultured in transwells, secreted trypsin-like proteolytic activity remained stable upon NOD2 stimulation but decreased under autophagy induction. Thapsigargin (Tg) stimulation a well-known ER-stress inducer, enhanced the apical release of trypsin-like activity in Caco2 cells. Activity-based probe assay identified a unique band at 33-KDa in ER-Stress-induced Caco-2 supernatants. This band showed specificity for Trypsin-3 in western blot. In UC patients, immunochemistry of colonic biopsies showed that Trypsin-3 was detectable mainly in epithelial cells, and up-regulated compared to biopsies from healthy controls and CD. Similarly, only UC patients displayed altered ER-stress with increased XBP1s mRNA levels. In Caco-2 cells, ER-Stress induction provoked increased paracellular permeability, CXCL8 release, antimicrobial peptides (AMP) (TFF-3 and HBD2), and mucins (MUC2) dysregulation. Serine protease inhibitor AEBSF inhibited Tg-induced increased permeability and AMP dysregulation, while CXCL8 increase was aggravated. In Caco-2, Tg-induced ER-Stress increased PAR-2 and -4 mRNA expression, PAR4 control levels were restored in the presence of AEBSF. ER-Stress-associated increased paracellular permeability was suppressed by PAR2 and/or -4 antagonist treatment, while CXCL8 was aggravated. Trypsin-3 did not induce ER stress in Caco2. Our data showed that in intestinal epithelial cells, ER-Stress increased trypsin-3 expression and trypsin proteolytic activity, which is responsible for altered barrier function and dysregulated AMP and mucin expressions. We identified PAR-2 and -4 activation as possible mechanisms by which ER-Stress contributed to epithelial pathophysiology. Trypsin-3 appears as a candidate protease overexpressed upon ER-Stress and in UC patients epithelium

The level of claudin-7 is reduced as an early event in colorectal carcinogenesis

<p>Abstract</p> <p>Background</p> <p>Compromised epithelial barriers are found in dysplastic tissue of the gastrointestinal tract. Claudins are transmembrane proteins important for tight junctions. Claudins regulate the paracellular transport and are crucial for maintaining a functional epithelial barrier. Down-regulation of the oncogenic serine protease, matriptase, induces leakiness in epithelial barriers both <it>in vivo </it>and <it>in vitro</it>. We found in an <it>in-silico </it>search tight co-regulation between <it>matriptase </it>and <it>claudin-7 </it>expression. We have previously shown that the <it>matriptase </it>expression level decreases during colorectal carcinogenesis. In the present study we investigated whether <it>claudin-7 </it>expression is likewise decreased during colorectal carcinogenesis, thereby causing or contributing to the compromised epithelial leakiness of dysplastic tissue.</p> <p>Methods</p> <p>The mRNA level of <it>claudin-7 </it>(CLDN7) was determined in samples from 18 healthy individuals, 100 individuals with dysplasia and 121 colorectal cancer patients using quantitative real time RT-PCR. In addition, immunohistochemical stainings were performed on colorectal adenomas and carcinomas, to confirm the mRNA findings.</p> <p>Results</p> <p>A 2.7-fold reduction in the <it>claudin-7 </it>mRNA level was found when comparing the biopsies from healthy individuals with the biopsies of carcinomas (p < 0.001). Reductions in the <it>claudin-7 </it>mRNA levels were also detected in mild/moderate dysplasia (p < 0.001), severe dysplasia (p < 0.01) and carcinomas (p < 0.01), compared to a control sample from the same individual. The decrease at mRNA level was confirmed at the protein level by immunohistochemical stainings.</p> <p>Conclusions</p> <p>Our results show that the <it>claudin-7 </it>mRNA level is decreased already as an early event in colorectal carcinogenesis, probably contributing to the compromised epithelial barrier in adenomas.</p

Early newborn care practices and the development and functionality of the human microbiome

Background: The protease family, a group of enzymes produced by the host and bacterial species, play important roles in maintaining the homeostasis of the intestinal barrier. The concentrations of major gastrointestinal proteases including; pepsin, trypsin, and chymotrypsin, have been measured in preterm neonates. However, their longitudinal development is not well described. This thesis aims to investigate the dynamics of proteases, relate activity to disease and bacterial colonisation and measure their impact in vitro in a neonatal cohort. Methods: Daily faecal and ileostomy samples were collected from birth until discharge or death in 66 preterm neonates born before 32 weeks’ gestation. Substrate and inhibitor-based assays were applied to profile total protease activity (ng/µL trypsin equivalent (TE)) in 20 control neonates, three surgically confirmed necrotising enterocolitis (NEC) cases, one spontaneous intestinal perforation (SIP) case, one intestinal atresia case, and three culture confirmed septicaemia cases. Postnatal colonisation of the intestinal tract was followed in 15 neonates by metataxonomic analysis. The proteolytic impact on the intestinal barrier was assessed in a Caco-2 cell model. Results: Control, NEC/SIP and septicaemia neonates were characterised by patterns of high and low proteolytic activities. Activity increased with postmenstrual age only in control neonates (p<0.05). Inhibitor analysis revealed faecal and ileostomy samples were characterised by serine and cysteine proteases. Colonisation was irregular and dominated by genera that were not influenced by postnatal age, mode of delivery or feeding regimen. Correlation analysis revealed positive associations between Clostridium sensu stricto and Corynebacterium genera and faecal and ileostomy proteolytic activity (p <0.05). Caco-2 cell exposure to samples with low protease activity (< 250 ng/µL TE) led to a greater decrease in transepithelial electrical resistance. Conclusions: Having established that proteases are present and active during the early neonatal period; this thesis provides the scope to continue investigating their associations with bacteria and their roles in neonatal diseases.Open Acces