scholarly journals miR-200b inhibits TNF-α-induced IL-8 secretion and tight junction disruption of intestinal epithelial cells in vitro

2017 ◽  
Vol 312 (2) ◽  
pp. G123-G132 ◽  
Author(s):  
Yujie Shen ◽  
Min Zhou ◽  
Junkai Yan ◽  
Zizhen Gong ◽  
Yongtao Xiao ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Light Chain ◽  
Myosin Light Chain Kinase ◽  
Myosin Light Chain ◽  
Intestinal Epithelial Cells ◽  
Paracellular Permeability ◽  
Intestinal Epithelial ◽  
Tnf Α ◽  
Epithelial Inflammation

Inflammatory bowel diseases (IBDs) are chronic, inflammatory disorders of the gastrointestinal tract with unclear etiologies. Intestinal epithelial cells (IECs), containing crypt and villus enterocytes, occupy a critical position in the pathogenesis of IBDs and are a major producer of immunoregulatory cytokines and a key component of the intact epithelial barrier. Previously, we have reported that miR-200b is involved in the progression of IBDs and might maintain the integrity of the intestinal epithelial barrier via reducing the loss of enterocytes. In this study, we further investigated the impact of miR-200b on intestinal epithelial inflammation and tight junctions in two distinct differentiated states of Caco-2 cells after TNF-α treatment. We demonstrated that TNF-α-enhanced IL-8 expression was decreased by microRNA (miR)-200b in undifferentiated IECs. Simultaneously, miR-200b could alleviate TNF-α-induced tight junction (TJ) disruption in well-differentiated IECs by reducing the reduction in the transepithelial electrical resistance (TEER), inhibiting the increase in paracellular permeability, and preventing the morphological redistribution of the TJ proteins claudin 1 and ZO-1. The expression levels of the JNK/c-Jun/AP-1 and myosin light chain kinase (MLCK)/phosphorylated myosin light chain (p-MLC) pathways were attenuated in undifferentiated and differentiated enterocytes, respectively. Furthermore, a dual-luciferase reporter gene detection system provided direct evidence that c-Jun and MLCK were the specific targets of miR-200b. Collectively, our results highlighted that miR-200b played a positive role in IECs via suppressing intestinal epithelial IL-8 secretion and attenuating TJ damage in vitro, which suggested that miR-200b might be a promising strategy for IBD therapy. NEW & NOTEWORTHY This was the first time that the inhibitory role of miR-200b on intestinal epithelial inflammation and paracellular permeability has been reported. Moreover, we further divided the intestinal epithelial cells (IECs) into two differentiated conditions and investigated the distinct impacts of miR-200b. Finally, we put forward and proved that myosin light chain kinase (MLCK) was a novel target of miR-200b.

scholarly journals Ca++-calmodulin-dependent phosphorylation of myosin, and its role in brush border contraction in vitro.

1982 ◽  
Vol 95 (3) ◽  
pp. 943-959 ◽  
Author(s):  
T C Keller ◽  
M S Mooseker
Keyword(s):  
Light Chain ◽  
Brush Border ◽  
Myosin Light Chain Kinase ◽  
Myosin Light Chain ◽  
Intestinal Epithelial Cells ◽  
Intestinal Epithelial ◽  
Anti Psychotic ◽  
Terminal Web ◽  
Brush Borders

We have reinvestigated the effects of Ca++ and ATP on brush borders isolated from intestinal epithelial cells. At 37 degrees C, Ca++ (1 microM) and ATP cause a dramatic contraction of brush border terminal webs, not a retraction of microvilli as previously reported (M. S. Mooseker, 1976, J. Cell Biol. 71:417-433). Terminal web contraction, which occurs over the course of 1-5 min at 37 degrees C, actively constricts brush borders at the level of their zonula adherens. Contraction requires ATP, is stimulated by Ca++ (1 microM), and occurs in both membrane-intact and demembranated brush borders. Ca++ -dependent-solation of microvillus cores requires a concentration of Ca++ slightly greater (10 microM) than that required for contraction. Under conditions in which brush borders contract, many proteins in the isolated brush borders become phosphorylated. However, the phosphorylation of only one of the brush border proteins, the 20,000 dalton (20-kdalton) light chain of brush border myosin (BBMLC20), is stimulated by Ca++. At 37 degrees C, BBMLC20 phosphorylation correlates directly with brush border contraction. Furthermore, both BBMLC20 phosphorylation and brush border contraction are inhibited by trifluoperazine, an anti-psychotic phenothiazine that inhibits calmodulin activity. These results indicate that Ca++ regulates brush border contractility in vitro by stimulating cytoskeleton-associated, Ca++- and calmodulin-dependent brush border myosin light chain kinase.

Mechanism of TNF-α modulation of Caco-2 intestinal epithelial tight junction barrier: role of myosin light-chain kinase protein expression

2005 ◽  
Vol 288 (3) ◽  
pp. G422-G430 ◽  
Author(s):  
Thomas Y. Ma ◽  
Michel A. Boivin ◽  
Dongmei Ye ◽  
Ali Pedram ◽  
Hamid M. Said
Keyword(s):  
Protein Expression ◽  
Tight Junction ◽  
Light Chain ◽  
Myosin Light Chain Kinase ◽  
Myosin Light Chain ◽  
Intestinal Inflammation ◽  
Intestinal Epithelial ◽  
Mrna Transcription ◽  
Tnf Α ◽  
Epithelial Tight Junction

TNF-α plays a central role in the intestinal inflammation of various inflammatory disorders including Crohn's disease (CD). TNF-α-induced increase in intestinal epithelial tight junction (TJ) permeability has been proposed as one of the proinflammatory mechanisms contributing to the intestinal inflammation. The intracellular mechanisms involved in the TNF-α-induced increase in intestinal TJ permeability remain unclear. The purpose of this study was to investigate the possibility that the TNF-α-induced increase in intestinal epithelial TJ permeability was regulated by myosin light-chain kinase (MLCK) protein expression, using an in vitro intestinal epithelial model system consisting of the filter-grown Caco-2 intestinal epithelial monolayers. TNF-α (10 ng/ml) produced a time-dependent increase in Caco-2 MLCK expression. The TNF-α increase in MLCK protein expression paralleled the increase in Caco-2 TJ permeability, and the inhibition of the TNF-α-induced MLCK expression (by cycloheximide) prevented the increase in Caco-2 TJ permeability, suggesting that MLCK expression may be required for the increase in Caco-2 TJ permeability. The TNF-α increase in MLCK protein expression was preceded by an increase in MLCK mRNA expression but not an alteration in MLCK protein degradation. Actinomycin-D prevented the TNF-α increase in MLCK mRNA expression and the subsequent increase in MLCK protein expression and Caco-2 TJ permeability, suggesting that the increase in MLCK mRNA transcription led to the increase in MLCK expression. The TNF-α increase in MLCK protein expression was also associated with an increase in Caco-2 MLCK activity. The cycloheximide inhibition of MLCK protein expression prevented the TNF-α increase in MLCK activity and Caco-2 TJ permeability. Moreover, inhibitors of MLCK, Mg2+-myosin ATPase, and metabolic energy prevented the TNF-α increase in Caco-2 TJ permeability, suggesting that the increase in MLCK activity was required for the TNF-α-induced opening of the Caco-2 TJ barrier. In conclusion, our results indicate for the first time that 1) the TNF-α increase in Caco-2 TJ permeability was mediated by an increase in MLCK protein expression, 2) the increase in MLCK protein expression was regulated by an increase in MLCK mRNA transcription, and 3) the increase in Caco-2 TJ permeability required MLCK protein expression-dependent increase in MLCK activity.

Osteoprotegerin production by human intestinal epithelial cells: a potential regulator of mucosal immune responses

2004 ◽  
Vol 287 (4) ◽  
pp. G836-G844 ◽  
Author(s):  
Karine Vidal ◽  
Patrick Serrant ◽  
Brigitte Schlosser ◽  
Peter van den Broek ◽  
Florence Lorget ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Immune Responses ◽  
Culture Supernatant ◽  
Intestinal Epithelial Cells ◽  
Biologically Active ◽  
Intestinal Epithelial ◽  
Ht29 Cells ◽  
Tnf Α ◽  
Important Regulator

Receptor activator of NF-κB (RANK) and its ligand (RANKL) are important members of the TNF receptor (TNFR) and TNF superfamilies, respectively. RANK is expressed on osteoclasts, T-lymphocytes, and dendritic cells, and its ligation with RANKL leads to cellular activation. However, another member of the TNFR family, osteoprotegerin (OPG), acts as a decoy receptor, binding to RANKL and preventing its interaction with RANK. Furthermore, OPG also binds TNF-related apoptosis-inducing ligand (TRAIL), an important regulator of cell survival. OPG is therefore an important regulator of bone metabolism and immune responses. Although intestinal epithelial cells (IEC) express some members of the TNF/TNFR superfamilies, the roles of OPG and RANKL in the intestinal mucosa has not been investigated. Here, we report that various human IEC lines constitutively express OPG mRNA and protein as well as mRNA for RANKL. Furthermore, human colonic epithelium constitutively expressed OPG, and this expression was increased in inflamed tissue. All of the IEC lines tested released OPG into the culture supernatant under standard culture conditions. Whereas TNF-α increased OPG protein secretion by HT29 cells, the cytokines IL-1β and IFN-γ had little, if any, effect. Furthermore, the culture supernatant from untreated HT29 cells abrogated TRAIL-induced inhibition of Jurkat T-cell proliferation and inhibited osteoclast activity in an in vitro model of bone resorption. Taken together, our data indicate that OPG is constitutively produced by IEC, could be upregulated by TNF-α, and is biologically active. Thus IEC-derived OPG may represent an important mucosal immunoregulatory factor and may be involved in bone physiology.

scholarly journals Prohibitin Inhibits Tumor Necrosis Factor alpha–induced Nuclear Factor-kappa B Nuclear Translocation via the Novel Mechanism of Decreasing Importin α3 Expression

2009 ◽  
Vol 20 (20) ◽  
pp. 4412-4423 ◽  
Author(s):  
Arianne L. Theiss ◽  
Aaron K. Jenkins ◽  
Ngozi I. Okoro ◽  
Jan-Michael A. Klapproth ◽  
Didier Merlin ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Intestinal Inflammation ◽  
Nuclear Translocation ◽  
Intestinal Epithelial Cells ◽  
Intestinal Epithelial ◽  
Necrosis Factor Alpha ◽  
Tnf Α ◽  
Factor Alpha

Expression of prohibitin 1 (PHB), a multifunctional protein in the cell, is decreased during inflammatory bowel disease (IBD). Little is known regarding the regulation and role of PHB during intestinal inflammation. We examined the effect of tumor necrosis factor alpha (TNF-α), a cytokine that plays a central role in the pathogenesis of IBD, on PHB expression and the effect of sustained PHB expression on TNF-α activation of nuclear factor-kappa B (NF-κB) and epithelial barrier dysfunction, two hallmarks of intestinal inflammation. We show that TNF-α decreased PHB protein and mRNA abundance in intestinal epithelial cells in vitro and in colon mucosa in vivo. Sustained expression of prohibitin in intestinal epithelial cells in vitro and in vivo (prohibitin transgenic mice, PHB TG) resulted in a marked decrease in TNF-α–induced nuclear translocation of the NF-κB protein p65, NF-κB/DNA binding, and NF-κB–mediated transcriptional activation despite robust IκB-α phosphorylation and degradation and increased cytosolic p65. Cells overexpressing PHB were protected from TNF-α–induced increased epithelial permeability. Expression of importin α3, a protein involved in p50/p65 nuclear import, was decreased in cells overexpressing PHB and in colon mucosa of PHB TG mice. Restoration of importin α3 levels sustained NF-κB activation by TNF-α during PHB transfection. These results suggest that PHB inhibits NF-κB nuclear translocation via a novel mechanism involving alteration of importin α3 levels. TNF-α decreases PHB expression in intestinal epithelial cells and restoration of PHB expression in these cells can protect against the deleterious effects of TNF-α and NF-κB on barrier function.

Myosin Light Chain Kinase Inhibition: Correction of Increased Intestinal Epithelial Permeability In Vitro

2007 ◽  
Vol 25 (6) ◽  
pp. 1377-1386 ◽  
Author(s):  
Linda M. Feighery ◽  
Sean W. Cochrane ◽  
Teresa Quinn ◽  
Alan W. Baird ◽  
Daniel O’Toole ◽  
...  
Keyword(s):  
Light Chain ◽  
Myosin Light Chain Kinase ◽  
Myosin Light Chain ◽  
Intestinal Epithelial ◽  
Epithelial Permeability ◽  
Kinase Inhibition
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