scholarly journals Mechanism of glucocorticoid regulation of the intestinal tight junction barrier

2007 ◽  
Vol 292 (2) ◽  
pp. G590-G598 ◽  
Author(s):  
Michel A. Boivin ◽  
Dongmei Ye ◽  
John C. Kennedy ◽  
Rana Al-Sadi ◽  
Chris Shepela ◽  
...  
Keyword(s):  
Crohn’S Disease ◽  
Crohn's Disease ◽  
Protein Expression ◽  
Tight Junction ◽  
Barrier Function ◽  
In Vitro Model ◽  
Intestinal Epithelial ◽  
Tnf Α ◽  
Vitro Model

A defective intestinal epithelial tight junction (TJ) barrier has been proposed as an important pathogenic factor contributing to the intestinal inflammation of Crohn's disease. Glucocorticoids are first-line therapeutic agents for the treatment of moderate to severe Crohn's disease. Glucocorticoid treatment has been shown to induce retightening of the intestinal TJ barrier defect in Crohn's disease patients. However, the mechanisms that mediate the glucocorticoid therapeutic action on intestinal TJ barrier function remain unknown. The aim of this study was to elucidate the mechanism of glucocorticoid modulation of the intestinal epithelial TJ barrier using an in vitro model system. Filter-grown Caco-2 intestinal epithelial cells were used as an in vitro model to examine the effects of glucocorticoids on basal intestinal epithelial TJ barrier function and on TNF-α-induced disruption of the TJ barrier. Glucocorticoids (prednisolone and dexamethasone) did not have a significant effect on baseline Caco-2 TJ barrier function but prevented the TNF-α-induced increase in Caco-2 TJ permeability. The glucocorticoid protective effect against the TNF-α-induced increase in Caco-2 TJ permeability required activation of the glucocorticoid receptor (GR) complex. The activation of the GR complex resulted in GR complex binding to the glucocorticoid response element (GRE) site on DNA and activation of a GR-responsive promoter. Glucocorticoids inhibited the TNF-α-induced increase in myosin light chain kinase (MLCK) protein expression, a key process mediating the TNF-α increase in intestinal TJ permeability. The glucocorticoid inhibition of the TNF-α-induced increase in MLCK protein expression was due to the binding of the GR complex to a GRE binding site on the MLCK promoter region suppressing the TNF-α-induced activation. Glucocorticoids inhibit the TNF-α-induced increase in Caco-2 TJ permeability. The prednisolone protective action was mediated by binding of activated GR complex to the GRE site on the MLCK promoter, suppressing the TNF-α-induced increase in MLCK gene activity, protein expression, and subsequent opening of the intestinal TJ barrier.

Enteroids Generated from Patients with Severe Inflammation in Crohn’s Disease Maintain Alterations of Junctional Proteins

2020 ◽  
Vol 14 (10) ◽  
pp. 1473-1487 ◽  
Author(s):  
Michael Meir ◽  
Jonas Salm ◽  
Christina Fey ◽  
Matthias Schweinlin ◽  
Catherine Kollmann ◽  
...  
Keyword(s):  
Crohn’S Disease ◽  
Crohn's Disease ◽  
Tight Junction ◽  
In Vitro Model ◽  
Transcriptional Level ◽  
Mrna Levels ◽  
Severe Inflammation ◽  
Junctional Proteins ◽  
Vitro Model

Abstract Background The mechanisms underlying loss of intestinal epithelial barrier [IEB] function in Crohn’s disease [CD] are poorly understood. We tested whether human enteroids generated from isolated intestinal crypts of CD patients serve as an appropriate in vitro model to analyse changes of IEB proteins observed in patients’ specimens. Methods Gut samples from CD patients and healthy individuals who underwent surgery were collected. Enteroids were generated from intestinal crypts and analyses of junctional proteins in comparison to full wall samples were performed. Results Histopathology confirmed the presence of CD and the extent of inflammation in intestinal full wall sections. As revealed by immunostaining and Western blot analysis, profound changes in expression patterns of tight junction, adherens junction and desmosomal proteins were observed in full wall specimens when CD was present. Unexpectedly, when enteroids were generated from specimens of CD patients with severe inflammation, alterations of most tight junction proteins and the majority of changes in desmosomal proteins but not E-cadherin were maintained under culture conditions. Importantly, these changes were maintained without any additional stimulation of cytokines. Interestingly, qRT-PCR demonstrated that mRNA levels of junctional proteins were not different when enteroids from CD patients were compared to enteroids from healthy controls. Conclusions These data indicate that enteroids generated from patients with severe inflammation in CD maintain some characteristics of intestinal barrier protein changes on a post-transcriptional level. The enteroid in vitro model represents an appropriate tool to gain further cellular and molecular insights into the pathogenesis of barrier dysfunction in CD.

TNF-α-induced increase in intestinal epithelial tight junction permeability requires NF-κB activation

2004 ◽  
Vol 286 (3) ◽  
pp. G367-G376 ◽  
Author(s):  
Thomas Y. Ma ◽  
Gary K. Iwamoto ◽  
Neil T. Hoa ◽  
Vimesh Akotia ◽  
Ali Pedram ◽  
...  
Keyword(s):  
Protein Expression ◽  
Tight Junction ◽  
Etiologic Factor ◽  
Intestinal Epithelial ◽  
Tnf Α ◽  
Abnormal Increase ◽  
Epithelial Tight Junction ◽  
Tight Junction Permeability ◽  
First Time

Crohn's disease (CD) patients have an abnormal increase in intestinal epithelial permeability. The defect in intestinal tight junction (TJ) barrier has been proposed as an important etiologic factor of CD. TNF-α increases intestinal TJ permeability. Because TNF-α levels are markedly increased in CD, TNF-α increase in intestinal TJ permeability could be a contributing factor of intestinal permeability defect in CD. Our purpose was to determine some of the intracellular mechanisms involved in TNF-α modulation of intestinal epithelial TJ permeability by using an in vitro intestinal epithelial system consisting of filter-grown Caco-2 monolayers. TNF-α produced a concentration- and time-dependent increase in Caco-2 TJ permeability. TNF-α-induced increase in Caco-2 TJ permeability correlated with Caco-2 NF-κB activation. Inhibition of TNF-α-induced NF-κB activation by selected NF-κB inhibitors, curcumin and triptolide, prevented the increase in Caco-2 TJ permeability, indicating that NF-κB activation was required for the TNF-α-induced increase in Caco-2 TJ permeability. This increase in Caco-2 TJ permeability was accompanied by down-regulation of zonula occludens (ZO)-1 proteins and alteration in junctional localization of ZO-1 proteins. TNF-α modulation of ZO-1 protein expression and junctional localization were also prevented by NF-κB inhibitors. TNF-α did not induce apoptosis in Caco-2 cells, suggesting that apoptosis was not the mechanism involved in TNF-α-induced increase in Caco-2 TJ permeability. These results demonstrate for the first time that TNF-α-induced increase in Caco-2 TJ permeability was mediated by NF-κB activation. The increase in permeability was associated with NF-κB-dependent downregulation of ZO-1 protein expression and alteration in junctional localization.

scholarly journals Unconjugated Bilirubin Modulates the Intestinal Epithelial Barrier Function in a Human-Derived In Vitro Model

2006 ◽  
Vol 60 (1) ◽  
pp. 30-33 ◽  
Author(s):  
Francesco Raimondi ◽  
Valeria Crivaro ◽  
Letizia Capasso ◽  
Luigi Maiuri ◽  
Pasquale Santoro ◽  
...  
Keyword(s):  
Barrier Function ◽  
In Vitro Model ◽  
Unconjugated Bilirubin ◽  
Epithelial Barrier ◽  
Intestinal Epithelial ◽  
Intestinal Epithelial Barrier ◽  
Epithelial Barrier Function ◽  
Vitro Model

Glucocorticoids regulate barrier function and claudin expression in intestinal epithelial cells via MKP-1

2014 ◽  
Vol 306 (3) ◽  
pp. G218-G228 ◽  
Author(s):  
Andreas Fischer ◽  
Markus Gluth ◽  
Friderike Weege ◽  
Ulrich-Frank Pape ◽  
Bertram Wiedenmann ◽  
...  
Keyword(s):  
Tight Junction ◽  
Barrier Function ◽  
Collagenous Colitis ◽  
Dependent Manner ◽  
Intestinal Epithelial ◽  
Barrier Dysfunction ◽  
Junction Protein ◽  
Bowel Diseases ◽  
Vitro Model

Barrier dysfunction is pivotal to the pathogenesis of inflammatory bowel diseases (IBD) and collagenous colitis. Glucocorticoids restore barrier function in Crohn's disease, but whether this reflects attenuated inflammation or an epithelial-specific action has not yet been addressed. Using filter-grown Caco-2 monolayers as an in vitro model of the intestinal epithelial barrier, we observed that glucocorticoids induced a time- and dose-dependent increase in transepithelial electrical resistance (TEER) in a glucocorticoid receptor-dependent manner without altering flux of larger solutes or changing principal tight junction architecture. This was accompanied by reduced paracellular cation flux, reduced expression of the pore-forming tight junction component claudin-2, and upregulation of the sealing tight junction protein claudin-4. In contrast, expression of occludin, claudin-1, -7, or -8 was not altered. Dexamethasone increased expression and activity of MAPK phosphatase-1 and inhibition of this phosphatase prevented the glucocorticoid-induced changes in TEER and claudin expression, whereas inhibiting p38 or MEK1/2 was not sufficient to replicate the glucocorticoid effects. Upon exposure to IFN-γ, TNF-α, or IL-1β, TEERs declined in dexamethasone-treated cells but remained consistently higher than in cells not receiving glucocorticoids. Treatment with IFN/TNF resulted in an upregulation of claudin-2 that was significantly attenuated by dexamethasone, whereas increased claudin-2 expression upon IL-1β stimulation was not affected by glucocorticoids. Taken together, barrier augmentation might represent a previously unrecognized mechanism of action, potentially contributing to the therapeutic efficacy of glucocorticoids in IBD and collagenous colitis.

scholarly journals MicroRNA-1297 suppressed the Akt/GSK3β signaling pathway and stimulated neural apoptosis in an in vivo sevoflurane exposure model

2021 ◽  
Vol 49 (4) ◽  
pp. 030006052098210
Author(s):  
Quan Wang ◽  
Jingcong Luo ◽  
Ruiqiang Sun ◽  
Jia Liu
Keyword(s):  
Protein Expression ◽  
Signaling Pathway ◽  
Neuronal Apoptosis ◽  
In Vitro Model ◽  
Nervous System Development ◽  
Exposure Model ◽  
Control Group ◽  
Pten Protein ◽  
Vitro Model

Objective Common inhalation anesthetics used for clinical anesthesia (such as sevoflurane) may induce nerve cell apoptosis during central nervous system development. Furthermore, anesthetics can produce cognitive impairments, such as learning and memory impairments, that continue into adulthood. However, the precise mechanism remains largely undefined. We aimed to determine the function of microRNA-1297 (miR-1297) in sevoflurane-induced neurotoxicity. Methods Reverse transcription-polymerase chain reaction assays were used to analyze miR-1297 expression in sevoflurane-exposed mice. MTT and lactate dehydrogenase (LDH) assays were used to measure cell growth, and neuronal apoptosis was analyzed using flow cytometry. Western blot analyses were used to measure PTEN, PI3K, Akt, and GSK3β protein expression. Results In sevoflurane-exposed mice, miR-1297 expression was up-regulated compared with the control group. MiR-1297 up-regulation led to neuronal apoptosis, inhibition of cell proliferation, and increased LDH activity in the in vitro model of sevoflurane exposure. MiR-1297 up-regulation also suppressed the Akt/GSK3β signaling pathway and induced PTEN protein expression in the in vitro model. PTEN inhibition (VO-Ohpic trihydrate) reduced PTEN protein expression and decreased the effects of miR-1297 down-regulation on neuronal apoptosis in the in vitro model. Conclusion Collectively, the results indicated that miR-1297 stimulates sevoflurane-induced neurotoxicity via the Akt/GSK3β signaling pathway by regulating PTEN expression.

scholarly journals Antagonism of Adherent Invasive E. coli LF82 With Human α-defensin 5 in the Follicle-associated Epithelium of Patients With Ileal Crohn’s Disease

2020 ◽  
Author(s):  
Lina Y Alkaissi ◽  
Martin E Winberg ◽  
Stéphanie DS Heil ◽  
Staffan Haapaniemi ◽  
Pär Myrelid ◽  
...  
Keyword(s):  
Crohn’S Disease ◽  
Crohn's Disease ◽  
Ex Vivo ◽  
Ussing Chambers ◽  
E Coli ◽  
Follicle Associated Epithelium ◽  
Vitro Model ◽  
Set Up

Abstract Background The first visible signs of Crohn’s disease (CD) are microscopic erosions over the follicle-associated epithelium (FAE). The aim of the study was to investigate the effects of human α-defensin 5 (HD5) on adherent-invasive Escherichia coli LF82 translocation and HD5 secretion after LF82 exposure in an in vitro model of human FAE and in human FAE ex vivo. Methods An in vitro FAE-model was set up by the coculture of Raji B cells and Caco-2-cl1 cells. Ileal FAE from patients with CD and controls were mounted in Ussing chambers. The effect of HD5 on LF82 translocation was studied by LF82 exposure to the cells or tissues with or without incubation with HD5. The HD5 secretion was measured in human FAE exposed to LF82 or Salmonella typhimurium. The HD5 levels were evaluated by immunofluorescence, immunoblotting, and ELISA. Results There was an increased LF82 translocation across the FAE-model compared with Caco-2-cl1 (P < 0.05). Incubation of cell/tissues with HD5 before LF82 exposure reduced bacterial passage in both models. Human FAE showed increased LF82 translocation in CD compared with controls and attenuated passage after incubation with sublethal HD5 in both CD and controls (P < 0.05). LF82 exposure resulted in a lower HD5 secretion in CD FAE compared with controls (P < 0.05), whereas Salmonella exposure caused equal secretion on CD and controls. There were significantly lower HD5 levels in CD tissues compared with controls. Conclusions Sublethal HD5 reduces the ability of LF82 to translocate through FAE. The HD5 is secreted less in CD in response to LF82, despite a normal response to Salmonella. This further implicates the integrated role of antimicrobial factors and barrier function in CD pathogenesis.

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