LRRC19 Promotes Permeability of the Gut Epithelial Barrier Through Degrading PKC-ζ and PKCι/λ to Reduce Expression of ZO1, ZO3, and Occludin

2021 ◽  
Author(s):  
Xiaomin Su ◽  
Jianmei Wei ◽  
Houbao Qi ◽  
Mengli Jin ◽  
Qianjing Zhang ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Fluorescein Isothiocyanate ◽  
Clinical Samples ◽  
Epithelial Barrier ◽  
Gut Barrier Function ◽  
Transmission Electron ◽  
Fluorescein Isothiocyanate Dextran ◽  
Pkc Ζ ◽  
Epithelium Cells

Abstract Background A dysfunctional gut epithelial barrier allows the augmented permeation of endotoxins, luminal antigens, and bacteria into the bloodstream, causing disease. The maintenance of gut epithelial barrier integrity may be regulated by multiple factors. Herein we analyze the role of leucine-rich repeat-containing protein 19 (LRRC19) in regulating the permeability of the gut epithelial barrier. Methods We utilized Lrrc19 knockout (KO) mice and clinical samples through transmission electron, intestinal permeability assay, Western blot, and immunofluorescence staining to characterize the role of LRRC19 in the permeability of the gut epithelial barrier. Results We found that LRRC19, which is expressed in gut epithelial cells, impairs gut barrier function. Transmission electron micrographs revealed a tighter junction and narrower gaps in the colon epithelium cells in LRRC19 KO mice. There were lower levels of serum lipopolysaccharide and 4 kDa-fluorescein isothiocyanate-dextran after gavage in LRRC19 KO mice than in wild-type mice. We found that LRRC19 could reduce the expression of zonula occludens (ZO)-1, ZO-3, and occludin in the colonic epithelial cells. The decreased expression of ZO-1, ZO-3, and occludin was dependent on degrading protein kinase C (PKC) ζ and PKCι/λ through K48 ubiquitination by LRRC19. The expression of LRRC19 was also negatively correlated with ZO-1, ZO-3, occludin, PKCζ, and PKCι/λ in human colorectal cancers. Conclusions The protein LRRC19 can promote the permeability of the gut epithelial barrier through degrading PKC ζ and PKCι/λ to reduce the expression of ZO-1, ZO-3, and occludin.

scholarly journals Porphyromonas gingivalis Impairs Oral Epithelial Barrier through Targeting GRHL2

2019 ◽  
Vol 98 (10) ◽  
pp. 1150-1158 ◽  
Author(s):  
W. Chen ◽  
A. Alshaikh ◽  
S. Kim ◽  
J. Kim ◽  
C. Chun ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Oral Mucosa ◽  
Porphyromonas Gingivalis ◽  
Oral Bacteria ◽  
Conditional Knockout ◽  
Epithelial Barrier ◽  
Wild Type ◽  
Oral Epithelial ◽  
Junction Proteins

Oral mucosa provides the first line of defense against a diverse array of environmental and microbial irritants by forming the barrier of epithelial cells interconnected by multiprotein tight junctions (TJ), adherens junctions, desmosomes, and gap junction complexes. Grainyhead-like 2 (GRHL2), an epithelial-specific transcription factor, may play a role in the formation of the mucosal epithelial barrier, as it regulates the expression of the junction proteins. The current study investigated the role of GRHL2 in the Porphyromonas gingivalis ( Pg)–induced impairment of epithelial barrier functions. Exposure of human oral keratinocytes (HOK-16B and OKF6 cells) to Pg or Pg-derived lipopolysaccharides ( Pg LPSs) led to rapid loss of endogenous GRHL2 and the junction proteins (e.g., zonula occludens, E-cadherin, claudins, and occludin). GRHL2 directly regulated the expression levels of the junction proteins and the epithelial permeability for small molecules (e.g., dextrans and Pg bacteria). To explore the functional role of GRHL2 in oral mucosal barrier, we used a Grhl2 conditional knockout (KO) mouse model, which allows for epithelial tissue-specific Grhl2 KO in an inducible manner. Grhl2 KO impaired the expression of the junction proteins at the junctional epithelium and increased the alveolar bone loss in the ligature-induced periodontitis model. Fluorescence in situ hybridization revealed increased epithelial penetration of oral bacteria in Grhl2 KO mice compared with the wild-type mice. Also, blood loadings of oral bacteria (e.g., Bacteroides, Bacillus, Firmicutes, β- proteobacteria, and Spirochetes) were significantly elevated in Grhl2 KO mice compared to the wild-type littermates. These data indicate that Pg bacteria may enhance paracellular penetration through oral mucosa in part by targeting the expression of GRHL2 in the oral epithelial cells, which then impairs the epithelial barrier by inhibition of junction protein expression, resulting in increased alveolar tissue destruction and systemic bacteremia.

scholarly journals Macropinocytosis as a Mechanism of Entry into Primary Human Urethral Epithelial Cells by Neisseria gonorrhoeae

2000 ◽  
Vol 68 (3) ◽  
pp. 1696-1699 ◽  
Author(s):  
Michael K. Zenni ◽  
Peter C. Giardina ◽  
Hillery A. Harvey ◽  
Jianqiang Shao ◽  
Margaret R. Ketterer ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Laser Scanning ◽  
Actin Polymerization ◽  
Kinase Inhibitors ◽  
Fluorescein Isothiocyanate ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Transmission Electron ◽  
Microscopy Analysis ◽  
Phosphoinositide 3 Kinase

ABSTRACT Gonococcal entry into primary human urethral epithelial cells (HUEC) can occur by macropinocytosis. Scanning and transmission electron microscopy revealed lamellipodia surrounding gonococci, and confocal laser scanning microscopy analysis showed organisms colocalized with M r 70,000 fluorescein isothiocyanate-labeled dextran within the cells. Phosphoinositide 3-kinase inhibitors and an actin polymerization inhibitor prevented macropinocytic entry of gonococci into HUEC.

scholarly journals Macrophages and Epithelial Cells Mutually Interact through NLRP3 to Clear Infection and Enhance the Gastrointestinal Barrier

Immuno ◽  
2021 ◽  
Vol 2 (1) ◽  
pp. 13-25
Author(s):  
Michael Bording-Jorgensen ◽  
Heather Armstrong ◽  
Madison Wickenberg ◽  
Paul LaPointe ◽  
Eytan Wine
Keyword(s):  
Epithelial Cells ◽  
Receptor Protein ◽  
Epithelial Barrier ◽  
Inflammasome Activation ◽  
Colonic Epithelial Cells ◽  
Epithelial Barrier Function ◽  
Barrier Integrity ◽  
Pathogen Control ◽  
Pathogen Clearance

Activation of the nod-like receptor protein 3 (NLRP3) leads to the release of the proinflammatory cytokine IL-1β, which then facilitates pathogen control by macrophages. The role of NLRPs in controlling infection of epithelial cells is not well understood. Our hypothesis was that activation of the NLRP3 inflammasome in colonic epithelial cells would promote macrophage-mediated epithelial recovery after infection with the pathogen Citrobacter rodentium. We devised a co-culture model using mouse colonic epithelial cells (CMT-93) and macrophages (J774A.1) during infection with C. rodentium. Inflammasome was activated using LPS and ATP and inhibited by YVAD. We assessed cytokine secretion (ELISA), macrophage recruitment and pathogen penetration (immunofluorescence), and epithelial barrier integrity (transepithelial electrical resistance). Macrophages were recruited to the apical membrane of epithelial cells, associated with tight junctions, promoted epithelial barrier recovery, and displaced C. rodentium. While NLRP3 was expressed in infected epithelial cells, IL-18 or IL-1β secretion remained unchanged. Supernatants from infected epithelial cells promoted infection clearance by macrophage; while this was inflammasome-independent, ATP significantly improved epithelial barrier recovery. The inflammasome appears to promote epithelial barrier function, independent of IL-18 and IL-1β secretion. Inflammasome activation in macrophages plays a dual role of promoting pathogen clearance and improving epithelial barrier integrity.

scholarly journals Inactivation of the Wnt/β-catenin signaling contributes to the epithelial barrier dysfunction induced by sodium oxalate in canine renal epithelial cells

2021 ◽  
Author(s):  
Yun Ji ◽  
Shuting Fang ◽  
Ying Yang ◽  
Zhenlong Wu
Keyword(s):  
Epithelial Cells ◽  
Barrier Function ◽  
Mdck Cells ◽  
Renal Stones ◽  
Sodium Oxalate ◽  
Epithelial Barrier ◽  
Barrier Dysfunction ◽  
Epithelial Barrier Function ◽  
Epithelial Barrier Dysfunction

Abstract Background Nephrolithiasis (also known as renal stones) is a common disease condition in companion animals, including dogs and cats. Dysfunction of renal tubular epithelial cells involves in the pathogenesis of renal stones. However, a functional role of Wnt/β-catenin signaling and its contribution to nephrolithiasis remains unknown. Results In the present study, we found that Mardin-Darby canine kidney (MDCK) cells treated with sodium oxalate resulted in reduced cell proliferation and migration, which was associated with the G0/G1 phase arrest of cell cycle progression. In addition, sodium oxalate exposure led to decreased transepithelial electrical resistance (TEER) and increased paracellular permeability. The deleterious effect of sodium oxalate on epithelial barrier function was related to decreased protein abundances of claudin-1, occludin, zonula occludens (ZO)-1, ZO-2 and ZO-3. Of note, protein levels of p-β-catenin (Ser552) in MDCK cells were repressed by sodium oxalate, indicating an inhibitory effect on the Wnt/β-catenin signaling. Intriguingly, SB216763, a GSK-3β inhibitor, enhanced the expression p-β-catenin (Ser552), and protected against epithelial barrier dysfunction in sodium oxalate-treated MDCK cells. Conclusion Taken together, our results revealed a critical role of Wnt/β-catenin signaling on the epithelial barrier function of MDCK cells. Activation of Wnt/β-catenin signaling might be an potentially therapeutic target for the treatment of renal stones in animals.

scholarly journals Functional role of kallikrein 5 and proteinase-activated receptor 2 in eosinophilic esophagitis

2020 ◽  
Vol 12 (545) ◽  
pp. eaaz7773 ◽  
Author(s):  
Nurit P. Azouz ◽  
Andrea M. Klingler ◽  
Purnima Pathre ◽  
John A. Besse ◽  
Netali Ben Baruch-Morgenstern ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Eosinophilic Esophagitis ◽  
Cytokine Production ◽  
Clinical Samples ◽  
Microbiome Composition ◽  
Peptidase Inhibitor ◽  
Esophageal Epithelial Cells ◽  
Protease Activated Receptor 2

Eosinophilic esophagitis (EoE) is a chronic, food antigen–driven, inflammatory disease of the esophagus and is associated with impaired barrier function. Evidence is emerging that loss of esophageal expression of the serine peptidase inhibitor, kazal type 7 (SPINK7), is an upstream event in EoE pathogenesis. Here, we provide evidence that loss of SPINK7 mediates its pro-EoE effects via kallikrein 5 (KLK5) and its substrate, protease-activated receptor 2 (PAR2). Overexpression of KLK5 in differentiated esophageal epithelial cells recapitulated the effect of SPINK7 gene silencing, including barrier impairment and loss of desmoglein-1 expression. Conversely, KLK5 deficiency attenuated allergen-induced esophageal protease activity, modified commensal microbiome composition, and attenuated eosinophilia in a murine model of EoE. Inhibition of PAR2 blunted the cytokine production associated with loss of SPINK7 in epithelial cells and attenuated the allergen-induced esophageal eosinophilia in vivo. Clinical samples substantiated dysregulated PAR2 expression in the esophagus of patients with EoE, and delivery of the clinically approved drug α1 antitrypsin (A1AT, a protease inhibitor) inhibited experimental EoE. These findings demonstrate a role for the balance between KLK5 and protease inhibitors in the esophagus and highlight EoE as a protease-mediated disease. We suggest that antagonizing KLK5 and/or PAR2 has potential to be therapeutic for EoE.

Endotoxin-induced ileal mucosal hyperpermeability in pigs: role of tissue acidosis

1994 ◽  
Vol 266 (4) ◽  
pp. G633-G646 ◽  
Author(s):  
A. L. Salzman ◽  
H. Wang ◽  
P. S. Wollert ◽  
T. J. Vandermeer ◽  
C. C. Compton ◽  
...  
Keyword(s):  
Fluorescein Isothiocyanate ◽  
Mucosal Permeability ◽  
Doppler Flowmetry ◽  
Tissue Ischemia ◽  
Mucosal Perfusion ◽  
Series Of Experiments ◽  
Tissue Acidosis ◽  
Fluorescein Isothiocyanate Dextran ◽  
Hypoxic Group

Administration of lipopolysaccharide (LPS) to experimental animals leads to diminished mesenteric perfusion, increased ileal mucosal [H+] , and increased gut epithelial permeability to hydrophilic solutes. We sought to determine whether these phenomena are causally related. Experiments were performed in anesthetized pigs. Permeability was assessed by measuring the plasma-to-lumen clearance of fluorescein isothiocyanate dextran (4,000 Da; FD-4) by a segment of ileum perfused with Ringer lactate solution. Mucosal perfusion (Qmuc) and [H4+] were estimated using laser-Doppler flowmetry and tonometry, respectively. In an initial series of experiments, we showed that mucosal permeability was linearly correlated with mucosal [H+] in animals subjected to graded degrees of mechanically induced mesenteric ischemia (n = 14, R2 = 0.58, P < 0.002) or injected with graded doses of LPS (n = 11, R2 = 0.93, P < 0.0001). In a second series of experiments, we induced mucosal acidosis in normal pigs by mechanical ventilation with either a hypoxic (n = 7) or a hypercapnic (n = 5) gas mixture. In both groups, ileal mucosal permeability to FD-4 increased significantly (P < 0.05), although transmesenteric release of lactate increased significantly only in the hypoxic group. Qmuc was unchanged in both groups. These data suggest that mucosal acidosis, even in the absence of tissue ischemia or hypoxia, increases intestinal permeability to a macromolecular hydrophilic solute. Tissue acidosis may be an important factor contributing to LPS-induced gut mucosal hyperpermeability.

scholarly journals Hypoxia increases transepithelial electrical conductance and reduces occludin at the plasma membrane in alveolar epithelial cells via PKC-ζ and PP2A pathway

2011 ◽  
Vol 300 (4) ◽  
pp. L569-L578 ◽  
Author(s):  
Juan Carlos Caraballo ◽  
Cecilia Yshii ◽  
Maria L. Butti ◽  
Whitney Westphal ◽  
Jennifer A. Borcherding ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Epithelial Cells ◽  
Electrical Conductance ◽  
Alveolar Epithelial Cells ◽  
Epithelial Barrier ◽  
Superoxide Dismutase 1 ◽  
Alveolar Epithelial ◽  
Hypoxic Environment ◽  
Phosphatase 2A ◽  
Pkc Ζ

During pulmonary edema, the alveolar space is exposed to a hypoxic environment. The integrity of the alveolar epithelial barrier is required for the reabsorption of alveolar fluid. Tight junctions (TJ) maintain the integrity of this barrier. We set out to determine whether hypoxia creates a dysfunctional alveolar epithelial barrier, evidenced by an increase in transepithelial electrical conductance (Gt), due to a decrease in the abundance of TJ proteins at the plasma membrane. Alveolar epithelial cells (AEC) exposed to mild hypoxia (Po2= 50 mmHg) for 30 and 60 min decreased occludin abundance at the plasma membrane and significantly increased Gt. Other cell adhesion molecules such as E-cadherin and claudins were not affected by hypoxia. AEC exposed to hypoxia increased superoxide, but not hydrogen peroxide (H2O2). Overexpression of superoxide dismutase 1 (SOD1) but not SOD2 prevented the hypoxia-induced Gtincrease and occludin reduction in AEC. Also, overexpression of catalase had a similar effect as SOD1, despite not detecting any increase in H2O2during hypoxia. Blocking PKC-ζ and protein phosphatase 2A (PP2A) prevented the hypoxia-induced occludin reduction at the plasma membrane and increase in Gt. In summary, we show that superoxide, PKC-ζ, and PP2A are involved in the hypoxia-induced increase in Gtand occludin reduction at the plasma membrane in AEC.

scholarly journals Interplay Between Microbiota, Toll-Like Receptors and Cytokines for the Maintenance of Epithelial Barrier Integrity

2021 ◽  
Vol 8 ◽  
Author(s):  
Iaroslav Semin ◽  
Justus Ninnemann ◽  
Marina Bondareva ◽  
Ilia Gimaev ◽  
Andrey A. Kruglov
Keyword(s):  
Epithelial Cells ◽  
Single Layer ◽  
Living Organism ◽  
The Body ◽  
Epithelial Barrier ◽  
Epithelial Tissue ◽  
Toll Like Receptors ◽  
Tissue Integrity ◽  
Healthy State

The intestinal tract is densely populated by microbiota consisting of various commensal microorganisms that are instrumental for the healthy state of the living organism. Such commensals generate various molecules that can be recognized by the Toll-like receptors of the immune system leading to the inflammation marked by strong upregulation of various proinflammatory cytokines, such as TNF, IL-6, and IL-1β. To prevent excessive inflammation, a single layer of constantly renewing, highly proliferating epithelial cells (IEC) provides proper segregation of such microorganisms from the body cavities. There are various triggers which facilitate the disturbance of the epithelial barrier which often leads to inflammation. However, the nature and duration of the stress may determine the state of the epithelial cells and their responses to cytokines. Here we discuss the role of the microbiota-TLR-cytokine axis in the maintenance of the epithelial tissue integrity. In particular, we highlight discrepancies in the function of TLR and cytokines in IEC barrier during acute or chronic inflammation and we suggest that intervention strategies should be applied based on the type of inflammation.

scholarly journals DOP51 Investigation on the role of Par4-associated cell polarity and associated barrier defects in inflammatory bowel diseases

2020 ◽  
Vol 14 (Supplement_1) ◽  
pp. S089-S089
Author(s):  
F Branchi ◽  
C Heldt ◽  
B Siegmund ◽  
M Schumann
Keyword(s):  
Epithelial Cells ◽  
Cell Polarity ◽  
Inflammatory Bowel Diseases ◽  
Laser Scanning Microscopy ◽  
Immunofluorescent Staining ◽  
Epithelial Barrier ◽  
Western Blots ◽  
Bowel Diseases ◽  
Inflammatory Bowel

Abstract Background In inflammatory bowel diseases (IBD), epithelial barrier defects occur as a consequence of chronic inflammation. Recent research suggested that cell polarity alterations may be upstream of barrier defects and additionally play a role in IBD-associated carcinogenesis (colitis-associated and small intestinal carcinoma). Par4 is a gene encoding a protein crucial in the development of cell polarity. LKB1, its human homologue, is mutated in Peutz–Jeghers syndrome (PJS), a genetic condition characterised by a higher risk of epithelial cancers. While the pivotal role of Par4/LKB1 in the development of epithelial cell polarity is established, its involvement in IBD-associated barrier defects and carcinogenesis is yet to be defined. Methods Endoscopic bowel mucosa samples from patients with Crohn’s disease, ulcerative colitis, PJS and controls were analysed to assess expression and localisation of Par4/LKB1. Cryosections were immunostained for Par4/LKB-1 along with a diverse set of markers of cell polarity and intercellular adhesion. The analysis was performed by confocal laser scanning microscopy in order to compare the expression of mucosal as well as the subcellular localisation of Par4/LKB1 in the gut mucosa. A quantitative analysis of protein expression with western blotting was performed as well. The function of Par4/LKB1 in intestinal epithelial cells (IEC) was evaluated by means of a Par4-deficient IEC model as well as in an IEC Par4-overexpression model. Moreover, a preliminary assessment of the possible role of Par4/LKB1-related polarity processes in IBD-associated carcinomas was performed through scanning genetic data from colitis-associated carcinomas for potential Par4-related mutations and altered Par4/LKB1 expression. Results The immunofluorescent staining allowed visualisation of intracellular expression of Par4 in epithelia from PJS, IBD patients and controls. In PJS-polyps, despite the alteration of regular tissue architecture typical of these lesions, the polarity of epithelial cells was maintained - contrary to control tissue, a punctate pattern of the Par4 staining was shown. In IBD tissue, no relevant differences in Par4 expression at confocal microscopy, as well as a quantitative assessment, were observed as compared with controls. No differences were observed in PJS or IBD samples as regards the expression of cell polarity markers such as Par3, CD71, crb3 or adhesion molecules such as ZO-1, e-cadherin, occludin, JAM-A. As regards the IEC model, we observed that Caco-2/BBe cells overexpressing Par4/LKB1 showed enhanced Par4/LKB1 cytosolic staining at immunofluorescence. In this model, a defective epithelial polarity and organisation on permeable supports could be observed in Caco2/BBe cysts in parallel with reduced expression of native LKB1 as seen in western blots. Preliminary analysis of data from our colitis-associated carcinomas database suggested various pathways potentially involving Par4/LKB1, yet no direct analysis of tissue samples for Par4-related mutations was performed yet. Conclusions Altered expression of Par4 in epithelial cells may influence the development of a functional epithelial barrier as suggested by IEC models. Par4/LKB1 as a known oncosuppressor gene is involved in a number of pathways potentially relevant for carcinogenesis in IBD; however, a direct connection between Par4-related alteration of the epithelial barrier and carcinogenesis is yet to be established.

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