Soluble factors fromLactobacillus GGactivate MAPKs and induce cytoprotective heat shock proteins in intestinal epithelial cells

2006 ◽  
Vol 290 (4) ◽  
pp. C1018-C1030 ◽  
Author(s):  
Yun Tao ◽  
Kenneth A. Drabik ◽  
Tonya S. Waypa ◽  
Mark W. Musch ◽  
John C. Alverdy ◽  
...  
Keyword(s):  
Signal Transduction ◽  
Heat Shock ◽  
Epithelial Cells ◽  
Map Kinases ◽  
Intestinal Epithelial Cells ◽  
Oxidant Stress ◽  
Signal Transduction Pathways ◽  
Clinical Effects ◽  
Dependent Manner ◽  
Intestinal Epithelial

Conditioned media from the probiotic Lactobacillus GG (LGG-CM) induce heat shock protein (Hsp) expression in intestinal epithelial cells. LGG-CM induces both Hsp25 and Hsp72 in a time- and concentration-dependent manner. These effects are mediated by a low-molecular-weight peptide that is acid and heat stable. DNA microarray experiments demonstrate that Hsp72 is one of the most highly upregulated genes in response to LGG-CM treatment. Real-time PCR and electrophoretic mobility shift assay confirm that regulation of Hsp induction is at least in part transcriptional in nature, involving heat shock factor-1. Although Hsps are not induced for hours after exposure, transient exposure to LGG-CM is sufficient to initiate the signal for Hsp induction, suggesting that signal transduction pathways may be involved. Experiments confirm that LGG-CM modulates the activity of certain signaling pathways in intestinal epithelial cells by activating MAP kinases. Inhibitors of p38 and JNK block the expression of Hsp72 normally induced by LGG-CM. Functional studies indicate that LGG-CM treatment of gut epithelial cells protects them from oxidant stress, perhaps by preserving cytoskeletal integrity. By inducing the expression of cytoprotective Hsps in gut epithelial cells, and by activating signal transduction pathways, the peptide product(s) secreted by LGG may contribute to the beneficial clinical effects attributed to this probiotic.

Flagellin is required for salmonella-induced expression of heat shock protein Hsp25 in intestinal epithelium

2008 ◽  
Vol 294 (3) ◽  
pp. G808-G818 ◽  
Author(s):  
Elaine O. Petrof ◽  
Mark W. Musch ◽  
Mae Ciancio ◽  
Jun Sun ◽  
Michael E. Hobert ◽  
...  
Keyword(s):  
Heat Shock ◽  
Epithelial Cells ◽  
Heat Shock Protein ◽  
P38 Mapk ◽  
Intestinal Epithelium ◽  
Intestinal Epithelial Cells ◽  
Oxidant Stress ◽  
Luciferase Reporter ◽  
Dependent Manner ◽  
Intestinal Epithelial

Flagellin is a bacterial protein responsible for activation of Toll-like receptor 5 (TLR5), which we hypothesize is involved in Salmonella's induction of cytoprotective heat shock proteins in intestinal epithelial cells. Flagellin induces the cytoprotective heat shock protein Hsp25 in different intestinal epithelial cell lines and in mouse intestine. Flagellin induces Hsp25 expression in a time-dependent manner in vitro. This effect is transcriptional, as confirmed by luciferase reporter assays and actinomycin D treatment. In addition, Hsp25 induction requires p38 MAPK activation and is only observed when flagellin is added to the basolateral side of polarized intestinal epithelial cells, consistent with the known location of TLR5. Flagellin-mediated Hsp25 induction is associated with increased protective effects against oxidant stress, an effect that is at least partially mediated by p38 MAPK. Use of small interfering RNA against Hsp25 demonstrates that flagellin-mediated protection against oxidant stress is to some degree mediated through Hsp25 induction. This suggests that, by protecting against oxidant injury, the induction of Hsp25 expression by flagellin may contribute to intestinal homeostasis. In a coculture cell model and in a mouse model of Salmonella enterica Serovar Typhimurium infection, not only does infection with wild-type and a flagellin-deletion mutant strain of Salmonella show that flagellin induces Hsp25 in vivo, but it also demonstrates that in the case of live Salmonella infection, flagellin serves as a major stimulus for the induction of Hsp25 expression. These data provide evidence that flagellin is required for Salmonella-mediated induction of Hsp25 expression in intestinal epithelium.

Heat induction of heat shock protein 25 requires cellular glutamine in intestinal epithelial cells

2006 ◽  
Vol 291 (2) ◽  
pp. C290-C299 ◽  
Author(s):  
Kittiporn Phanvijhitsiri ◽  
Mark W. Musch ◽  
Mark J. Ropeleski ◽  
Eugene B. Chang
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Heat Shock ◽  
Epithelial Cells ◽  
Heat Shock Protein ◽  
Cellular Response ◽  
Intestinal Epithelial Cells ◽  
Dependent Manner ◽  
Intestinal Epithelial ◽  
Heat Induction

Glutamine is considered a nonessential amino acid; however, it becomes conditionally essential during critical illness when consumption exceeds production. Glutamine may modulate the heat shock/stress response, an important adaptive cellular response for survival. Glutamine increases heat induction of heat shock protein (Hsp) 25 in both intestinal epithelial cells (IEC-18) and mesenchymal NIH/3T3 cells, an effect that is neither glucose nor serum dependent. Neither arginine, histidine, proline, leucine, asparagine, nor tyrosine acts as physiological substitutes for glutamine for heat induction of Hsp25. The lack of effect of these amino acids was not caused by deficient transport, although some amino acids, including glutamate (a major direct metabolite of glutamine), were transported poorly by IEC-18 cells. Glutamate uptake could be augmented in a concentration- and time-dependent manner by increasing either media concentration and/or duration of exposure. Under these conditions, glutamate promoted heat induction of Hsp25, albeit not as efficiently as glutamine. Further evidence for the role of glutamine conversion to glutamate was obtained with the glutaminase inhibitor 6-diazo-5-oxo-l-norleucine (DON), which inhibited the effect of glutamine on heat-induced Hsp25. DON inhibited phosphate-dependent glutaminase by 75% after 3 h, decreasing cell glutamate. Increased glutamine/glutamate conversion to glutathione was not involved, since the glutathione synthesis inhibitor, buthionine sulfoximine, did not block glutamine’s effect on heat induction of Hsp25. A large drop in ATP levels did not appear to account for the diminished Hsp25 induction during glutamine deficiency. In summary, glutamine is an important amino acid, and its requirement for heat-induced Hsp25 supports a role for glutamine supplementation to optimize cellular responses to pathophysiological stress.

scholarly journals Long Term Endocrine Regulation of Nucleoside Transporters in Rat Intestinal Epithelial Cells

2004 ◽  
Vol 124 (5) ◽  
pp. 505-512 ◽  
Author(s):  
Ivette Aymerich ◽  
Marçal Pastor-Anglada ◽  
F. Javier Casado
Keyword(s):  
Signal Transduction ◽  
Epithelial Cells ◽  
Intestinal Epithelial Cells ◽  
Basolateral Membrane ◽  
Nucleoside Transporters ◽  
Nucleoside Transport ◽  
Signal Transduction Pathways ◽  
Mrna Levels ◽  
Endocrine Regulation ◽  
Intestinal Epithelial

We studied the regulation of nucleoside transporters in intestinal epithelial cells upon exposure to either differentiating or proliferative agents. Rat intestinal epithelial cells (line IEC-6) were incubated in the presence of differentiating (glucocorticoids) or proliferative (EGF and TGF-α) agents. Nucleoside uptake rates and nucleoside transporter protein and mRNA levels were assessed. The signal transduction pathways used by the proliferative stimuli were analyzed. We found that glucocorticoids induce an increase in sodium-dependent, concentrative nucleoside transport rates and in protein and mRNA levels of both rCNT2 and rCNT1, with negligible effects on the equilibrative transporters. EGF and TGF-α induce an increase in the equilibrative transport rate, mostly accounted for by an increase in rENT1 activity and mRNA levels, rENT2 mRNA levels remaining unaltered. This effect is mimicked by another proliferative stimulus that functions as an in vitro model of epithelial wounding. Here, rENT1 activity and mRNA levels are also increased, although the signal transduction pathways used by the two stimuli are different. We concluded that differentiation of rat intestinal epithelial cells is accompanied by increased mature enterocyte features, such as concentrative nucleoside transport (located at the brush border membrane of the enterocyte), thus preparing the cell for its ultimate absorptive function. A proliferative stimulus induces the equilibrative nucleoside activities (mostly through ENT1) known to be located at the basolateral membrane, allowing the uptake of nucleosides from the bloodstream for the increased demands of the proliferating cell.

scholarly journals PepT1 mediates transport of the proinflammatory bacterial tripeptide l-Ala-γ-d-Glu-meso-DAP in intestinal epithelial cells

2010 ◽  
Vol 299 (3) ◽  
pp. G687-G696 ◽  
Author(s):  
Guillaume Dalmasso ◽  
Hang Thi Thu Nguyen ◽  
Laetitia Charrier-Hisamuddin ◽  
Yutao Yan ◽  
Hamed Laroui ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Map Kinases ◽  
Intestinal Inflammation ◽  
Intestinal Epithelial Cells ◽  
Breakdown Product ◽  
Specific Substrate ◽  
Dependent Manner ◽  
Intestinal Epithelial ◽  
Proinflammatory Response ◽  
Chemokine Production

PepT1 is a di/tripeptide transporter highly expressed in the small intestine, but poorly or not expressed in the colon. However, during chronic inflammation, such as inflammatory bowel disease, PepT1 expression is induced in the colon. Commensal bacteria that colonize the human colon produce a large amount of di/tripeptides. To date, two bacterial peptides ( N-formylmethionyl-leucyl-phenylalanine and muramyl dipeptide) have been identified as substrates of PepT1. We hypothesized that the proinflammatory tripeptide l-Ala-γ-d-Glu- meso-DAP (Tri-DAP), a breakdown product of bacterial peptidoglycan, is transported into intestinal epithelial cells via PepT1. We found that uptake of glycine-sarcosine, a specific substrate of PepT1, in intestinal epithelial Caco2-BBE cells was inhibited by Tri-DAP in a dose-dependent manner. Tri-DAP induced activation of NF-κB and MAP kinases, consequently leading to production of the proinflammatory cytokine interleukin-8. Tri-DAP-induced inflammatory response in Caco2-BBE cells was significantly suppressed by silencing of PepT1 expression by using PepT1-shRNAs in a tetracycline-regulated expression ( Tet-off) system. Colonic epithelial HT29-Cl.19A cells, which do not express PepT1 under basal condition, were mostly insensitive to Tri-DAP-induced inflammation. However, HT29-Cl.19A cells exhibited proinflammatory response to Tri-DAP upon stable transfection with a plasmid encoding PepT1. Accordingly, Tri-DAP significantly increased keratinocyte-derived chemokine production in colonic tissues from transgenic mice expressing PepT1 in intestinal epithelial cells. Finally, Tri-DAP induced a significant drop in intracellular pH in intestinal epithelial cells expressing PepT1, but not in cells that did not express PepT1. Our data collectively support the classification of Tri-DAP as a novel substrate of PepT1. Given that PepT1 is highly expressed in the colon during inflammation, PepT1-mediated Tri-DAP transport may occur more effectively during such conditions, further contributing to intestinal inflammation.

scholarly journals Positive cross talk between protein kinase D and β-catenin in intestinal epithelial cells: impact on β-catenin nuclear localization and phosphorylation at Ser552

2016 ◽  
Vol 310 (7) ◽  
pp. C542-C557 ◽  
Author(s):  
Jia Wang ◽  
Liang Han ◽  
James Sinnett-Smith ◽  
Li-Li Han ◽  
Jan V. Stevens ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Epithelial Cells ◽  
Transgenic Mice ◽  
Nuclear Localization ◽  
Cross Talk ◽  
Intestinal Epithelial Cells ◽  
Dependent Manner ◽  
Intestinal Epithelial ◽  
Ang Ii ◽  
Potent Inducer

Given the fundamental role of β-catenin signaling in intestinal epithelial cell proliferation and the growth-promoting function of protein kinase D1 (PKD1) in these cells, we hypothesized that PKDs mediate cross talk with β-catenin signaling. The results presented here provide several lines of evidence supporting this hypothesis. We found that stimulation of intestinal epithelial IEC-18 cells with the G protein-coupled receptor (GPCR) agonist angiotensin II (ANG II), a potent inducer of PKD activation, promoted endogenous β-catenin nuclear localization in a time-dependent manner. A significant increase was evident within 1 h of ANG II stimulation ( P < 0.01), peaked at 4 h ( P < 0.001), and declined afterwards. GPCR stimulation also induced a marked increase in β-catenin-regulated genes and phosphorylation at Ser552 in intestinal epithelial cells. Exposure to preferential inhibitors of the PKD family (CRT006610 or kb NB 142-70) or knockdown of the isoforms of the PKD family prevented the increase in β-catenin nuclear localization and phosphorylation at Ser552 in response to ANG II. GPCR stimulation also induced the formation of a complex between PKD1 and β-catenin, as shown by coimmunoprecipitation that depended on PKD1 catalytic activation, as it was abrogated by cell treatment with PKD family inhibitors. Using transgenic mice that express elevated PKD1 protein in the intestinal epithelium, we detected a marked increase in the localization of β-catenin in the nucleus of crypt epithelial cells in the ileum of PKD1 transgenic mice, compared with nontransgenic littermates. Collectively, our results identify a novel cross talk between PKD and β-catenin in intestinal epithelial cells, both in vitro and in vivo.

scholarly journals Lipid rafts mediate internalization of β1-integrin in migrating intestinal epithelial cells

2008 ◽  
Vol 295 (5) ◽  
pp. G965-G976 ◽  
Author(s):  
Elena V. Vassilieva ◽  
Kirsten Gerner-Smidt ◽  
Andrei I. Ivanov ◽  
Asma Nusrat
Keyword(s):  
Epithelial Cells ◽  
Lipid Rafts ◽  
Lipid Raft ◽  
Intestinal Epithelial Cells ◽  
Focal Adhesions ◽  
Endocytic Pathway ◽  
Dependent Manner ◽  
Intestinal Epithelial ◽  
Caveolin 1 ◽  
Cell Matrix

Intestinal mucosal inflammation is associated with epithelial wounds that rapidly reseal by migration of intestinal epithelial cells (IECs). Cell migration involves cycles of cell-matrix adhesion/deadhesion that is mediated by dynamic turnover (assembly and disassembly) of integrin-based focal adhesions. Integrin endocytosis appears to be critical for deadhesion of motile cells. However, mechanisms of integrin internalization during remodeling of focal adhesions of migrating IECs are not understood. This study was designed to define the endocytic pathway that mediates internalization of β1-integrin in migrating model IECs. We observed that, in SK-CO15 and T84 colonic epithelial cells, β1-integrin is internalized in a dynamin-dependent manner. Pharmacological inhibition of clathrin-mediated endocytosis or macropinocytosis and small-interfering RNA (siRNA)-mediated knock down of clathrin did not prevent β1-integrin internalization. However, β1-integrin internalization was inhibited following cholesterol extraction and after overexpression of lipid raft protein, caveolin-1. Furthermore, internalized β1-integrin colocalized with the lipid rafts marker cholera toxin, and siRNA-mediated knockdown of caveolin-1 and flotillin-1/2 increased β1-integrin endocytosis. Our data suggest that, in migrating IEC, β1-integrin is internalized via a dynamin-dependent lipid raft-mediated pathway. Such endocytosis is likely to be important for disassembly of integrin-based cell-matrix adhesions and therefore in regulating IEC migration and wound closure.

Comparison of gene expression and activation of transcription factors in organoid-derived monolayer intestinal epithelial cells and organoids

2021 ◽  
Author(s):  
Yu Takahashi ◽  
Yu Inoue ◽  
Keitaro Kuze ◽  
Shintaro Sato ◽  
Makoto Shimizu ◽  
...  
Keyword(s):  
Gene Expression ◽  
Epithelial Cells ◽  
Gene Expression Regulation ◽  
Intestinal Epithelial Cells ◽  
Three Dimensional ◽  
Culture Conditions ◽  
Dependent Manner ◽  
Intestinal Epithelial

Abstract Intestinal organoids better represent in vivo intestinal properties than conventionally used established cell lines in vitro. However, they are maintained in three-dimensional culture conditions that may be accompanied by handling complexities. We characterized the properties of human organoid-derived two-dimensionally cultured intestinal epithelial cells (IECs) compared with those of their parental organoids. We found that the expression of several intestinal markers and functional genes were indistinguishable between monolayer IECs and organoids. We further confirmed that their specific ligands equally activate intestinal ligand-activated transcriptional regulators in a dose-dependent manner. The results suggest that culture conditions do not significantly influence the fundamental properties of monolayer IECs originating from organoids, at least from the perspective of gene expression regulation. This will enable their use as novel biological tools to investigate the physiological functions of the human intestine.

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