Conditionally Immortalized Intestinal Epithelial Cells: A New Model for Studying Intestinal Epithelial Cell Turnover

Endothelin (ET), a vasoconstrictive peptide, is known to have a variety of biological actions. Although ET is released by vascular endothelial cells, other cell populations also have been reported to synthesize and release ET. In this study, we examined whether ET is synthesized by intestinal epithelial cells and whether it affects induction of epithelial cell proliferation by interleukin-2 (IL-2). Subconfluent monolayers of intestinal epithelial cells (IEC-6 and IEC-18) were maintained in serum-free medium before addition of rat IL-2. Both IEC-6 and IEC-18 cells released ET-1 into the medium under unstimulated conditions, as determined by a sandwich ELISA. IL-2 significantly enhanced ET-1 release in a time-dependent manner. ET-3 was not detectable in the culture media of either cell line. Expression of ET-1 and ET-3 mRNA in epithelial cells was assessed by competitive PCR. Both cell lines were shown to express ET-1 mRNA, but no ET-3 mRNA was detected. IL-2 treatment enhanced ET-1 mRNA expression by both IEC-6 and IEC-18 cells. Both cell lines also expressed mRNA for ETA and ETB receptor subtypes. When cell proliferation was assessed, exogenous ET-1 induced a slight proliferative response in both types of cells that was consistent and significant at low ET-1 concentrations; cell growth was inhibited at a higher concentration (10−7M). IL-2 produced a significant proliferative response in both cell lines. However, the addition of ET-1 (10−7 M) to culture media attenuated the IL-2-induced increase in cell proliferation. ETA-receptor antagonists significantly enhanced cellular proliferation, suggesting involvement of the ETA receptor in modulation of IL-2-induced intestinal epithelial cell growth.

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Rapid protein kinase D1 signaling promotes migration of intestinal epithelial cells

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00025.2012 ◽

2012 ◽

Vol 303 (3) ◽

pp. G356-G366 ◽

Cited By ~ 10

Author(s):

Steven H. Young ◽

Nora Rozengurt ◽

James Sinnett-Smith ◽

Enrique Rozengurt

Keyword(s):

Protein Kinase ◽

Epithelial Cell ◽

Epithelial Cells ◽

Transgenic Mice ◽

Intestinal Epithelial Cell ◽

Intestinal Epithelial Cells ◽

Intestinal Epithelial ◽

Protein Kinase D1

We have examined the role of protein kinase D1 (PKD1) signaling in intestinal epithelial cell migration. Wounding monolayer cultures of intestinal epithelial cell line IEC-18 or IEC-6 induced rapid PKD1 activation in the cells immediately adjacent to the wound edge, as judged by immunofluorescence microscopy with an antibody that detects the phosphorylated state of PKD1 at Ser916, an autophosphorylation site. An increase in PKD1 phosphorylation at Ser916 was evident as early as 45 s after wounding, reached a maximum after 3 min, and persisted for ≥15 min. PKD1 autophosphorylation at Ser916 was prevented by the PKD family inhibitors kb NB 142-70 and CRT0066101. A kb NB 142-70-sensitive increase in PKD autophosphorylation was also elicited by wounding IEC-6 cells. Using in vitro kinase assays after PKD1 immunoprecipitation, we corroborated that wounding IEC-18 cells induced rapid PKD1 catalytic activation. Further results indicate that PKD1 signaling is required to promote migration of intestinal epithelial cells into the denuded area of the wound. Specifically, treatment with kb NB 142-70 or small interfering RNAs targeting PKD1 markedly reduced wound-induced migration in IEC-18 cells. To test whether PKD1 promotes migration of intestinal epithelial cells in vivo, we used transgenic mice that express elevated PKD1 protein in the small intestinal epithelium. Enterocyte migration was markedly increased in the PKD1 transgenic mice. These results demonstrate that PKD1 activation is one of the early events initiated by wounding a monolayer of intestinal epithelial cells and indicate that PKD1 signaling promotes the migration of these cells in vitro and in vivo.

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Schisandrin A protects intestinal cells from mycophenolic acid-induced cytotoxicity and oxidative damage

10.21203/rs.3.rs-767385/v1 ◽

2021 ◽

Author(s):

Yiyun Deng ◽

Zhe Zhang ◽

Yuanyuan Hong ◽

Lijuan Feng ◽

Yong Su ◽

...

Keyword(s):

Oxidative Stress ◽

Epithelial Cell ◽

Epithelial Cells ◽

Oxidative Damage ◽

Mycophenolic Acid ◽

Cell Apoptosis ◽

Intestinal Epithelial Cell ◽

Intestinal Epithelial Cells ◽

Mapk Signaling ◽

Intestinal Epithelial

Abstract Objectives: The gastrointestinal side effects of mycophenolic acid affect its efficacy in kidney transplant patients, which may be due to its toxicity to the intestinal epithelial mechanical barrier, including intestinal epithelial cell apoptosis and destruction of tight junctions. The toxicity mechanism of mycophenolic acid is related to oxidative stress-mediated the activation of mitogen-activated protein kinases (MAP K). Schisandrin A (Sch A), one of the main active components of the Schisandra chinensis, can protects intestinal epithelial cells from deoxynivalenol-induced cytotoxicity and oxidative damage by antioxidant effects. The aim of this study was to investigate the protective effect and potential mechanism of Sch A on mycophenolic acid-induced damage in intestinal epithelial cell. Methods: Caco-2 cells monolayers were treated with mycophenolic acid (10µM) and/or Sch A (10, 20 and 40µM) at 37°C for 24h, and cell viability was measured by MTT; Western blot and immunofluorescence were used to detect the expression of relevant proteins. Intracellular ROS and apoptosis were measured by flow cytometry, and malondialdehyde (MDA) and superoxide dismutase (SOD) levels were measured by kits. Results: The results showed that Sch A significantly reversed the mycophenolic acid-induced cell viability reduction, restored the expression of tight junction protein ZO-1, occludin and reduced cell apoptosis. In addition, Sch A inhibited mycophenolic acid-mediated MAPK activation and reactive oxygen species (ROS) increase. Conclusions: Sch A protected intestinal epithelial cells from mycophenolic acid intestinal toxicity, at least in part, by reducing oxidative stress and inhibiting MAPK signaling pathway. Conclusions: Sch A protected intestinal epithelial cells from mycophenolic acid intestinal toxicity, at least in part, by reducing oxidative stress and inhibiting MAPK signaling pathway.

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PSIX-17 Investigating the biological activities of sodium cellobionate produced from cellulosic biomass

Journal of Animal Science ◽

10.1093/jas/skaa054.330 ◽

2020 ◽

Vol 98 (Supplement_3) ◽

pp. 187-187

Author(s):

Lauren Kovanda ◽

Zhiliang Fan ◽

Xunde Li ◽

Yanhong Liu

Keyword(s):

Cell Proliferation ◽

Antimicrobial Activity ◽

Epithelial Cell ◽

Epithelial Cells ◽

Intestinal Epithelial Cell ◽

Intestinal Epithelial Cells ◽

Biological Activities ◽

Radical Scavenging ◽

Intestinal Epithelial

Abstract A novel method has been developed to easily hydrolyze cellulose to sodium cellobionate in a filamentous fungas, Neurospora crassa. The objectives of this experiment were to investigate the in vitro biological activities of sodium cellobionate. Antioxidant activity was evaluated with 3 chemical-based assays, including DPPH radical scavenging assay (DPPH), Trolox equivalent antioxidant capacity assay (TEAC), and ferric reducing antioxidant power assay (FRAP). Antimicrobial activity was determined as minimum inhibitory concentration (MIC) that prevented growth of tested bacteria, including four gram-negative bacteria (Escherichia coli F18 and ATCC 25922, and Salmonella Typhimurium ATCC 14028 and a wild strain isolated from cull diary cows in California) and one gram-positive bacteria (Enterococcus faecalis ATCC 29212). Anti-inflammatory activity was tested by analyzing TNF-α production with porcine alveolar macrophages that were challenged with lipopolysaccharide. A porcine intestinal epithelial cell line, IPEC-J2, was also used to test the effects of cellobionate on cell proliferation of intestinal epithelial cells. The tested doses of sodium cellobionate were 0, 0.04, 0.20, 1.00, 2.00, 4.00, 20.00, and 40.00 mg/mL. All assays were performed with over 6 replicates, except that MIC assays were performed as triplicate. All data were analyzed by PROC MIXED of SAS. Sodium cellobionate did not have radical scavenging capacity, but had weak FRAP (9.68 μM L-Cysteine equivalent) and TEAC (69% reduction) at the dose of 40 mg/mL. MIC results revealed that sodium cellobionate did not inhibit the growth of all tested bacteria, indicating it does not have antimicrobial activity within the range of tested doses. Sodium cellobionate did not exhibit anti-inflammatory activities, but significantly enhanced (P < 0.05) intestinal epithelial cell proliferation in vitro by 24.00%, 39.64%, and 25.98% when the doses were 1.00, 2.00, and 4.00 mg/mL, respectively. Results of this experiment indicate that cellobionate has limited biological activities in vitro, except that this biomass product could strongly stimulate the proliferation of intestinal epithelial cells. Future research will focus on the potential impacts of sodium cellobionate on intestinal physiology in vivo.

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Iron chelation acutely stimulates fetal human intestinal cell production of IL-6 and VEGF while decreasing HGF: the roles of p38, ERK, and JNK MAPK signaling

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00502.2006 ◽

2007 ◽

Vol 292 (4) ◽

pp. G958-G963 ◽

Cited By ~ 17

Author(s):

Troy A. Markel ◽

Paul R. Crisostomo ◽

Meijing Wang ◽

Christine M. Herring ◽

Tim Lahm ◽

...

Keyword(s):

Epithelial Cell ◽

Epithelial Cells ◽

P38 Mapk ◽

Intestinal Epithelial Cell ◽

Intestinal Epithelial Cells ◽

Iron Chelation ◽

Intestinal Epithelial ◽

Cell Production ◽

Human Intestinal Epithelial Cells ◽

Tnf Α

Bacteria have developed mechanisms to sequester host iron via chelators such as deferoxamine (DFO). Interestingly, DFO has been shown to stimulate acute intestinal epithelial cell inflammatory cytokine production in the absence of bacteria; however, this mechanism has not been elucidated. Intestinal epithelial cell production of IL-6 and TNF-α is elevated in various gastrointestinal pathologies, including acute intestinal ischemia. Similarly, VEGF and HGF are essential to intestinal epithelial cell integrity. Therapeutic strategies that decrease IL-6 and TNF-α while increasing VEGF and HGF therefore have theoretical appeal. We hypothesized that 1) fetal human intestinal epithelial cells acutely produce increased IL-6, TNF-α, VEGF, and HGF during iron chelation and 2) the MAPK pathway mediates these effects. Fetal human intestinal epithelial cells were stimulated by iron chelation (1 mM DFO) with and without p38 MAPK, ERK, or JNK inhibition. Supernatants were harvested after 24 h of incubation, and IL-6, TNF-α, VEGF, and HGF levels were quantified by ELISA. Activation of MAPK pathways was confirmed by Western blot analysis. DFO stimulation resulted in a significant increase in epithelial cell IL-6 and VEGF production while yielding a decrease in HGF production ( P < 0.05). Unexpectedly, TNF-α was not detectable. p38 MAPK, ERK, and JNK inhibition significantly decreased IL-6, VEGF, and HGF production ( P < 0.05). In conclusion, DFO acutely increases fetal human intestinal epithelial cell IL-6 and VEGF expression while causing an unexpected decrease in HGF expression and no detectable TNF-α production. Furthermore, chelator-induced intestinal epithelial cell cytokine expression depends on p38, ERK, and JNK MAPK pathways.

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Lactobacillus acidophilus Induces Cytokine and Chemokine Production via NF-κB and p38 Mitogen-Activated Protein Kinase Signaling Pathways in Intestinal Epithelial Cells

Clinical and Vaccine Immunology ◽

10.1128/cvi.05617-11 ◽

2012 ◽

Vol 19 (4) ◽

pp. 603-608 ◽

Cited By ~ 24

Author(s):

Yujun Jiang ◽

Xuena Lü ◽

Chaoxin Man ◽

Linlin Han ◽

Yi Shan ◽

...

Keyword(s):

Epithelial Cell ◽

Epithelial Cells ◽

Cell Lines ◽

Intestinal Epithelial Cell ◽

Intestinal Epithelial Cells ◽

Intestinal Epithelial ◽

Chemokine Production ◽

Content Type ◽

Cytokines And Chemokines ◽

Epithelial Cell Lines

ABSTRACTIntestinal epithelial cells can respond to certain bacteria by producing an array of cytokines and chemokines which are associated with host immune responses.Lactobacillus acidophilusNCFM is a characterized probiotic, originally isolated from human feces. This study aimed to test the ability ofL. acidophilusNCFM to stimulate cytokine and chemokine production in intestinal epithelial cells and to elucidate the mechanisms involved in their upregulation. In experiments using intestinal epithelial cell lines and mouse models, we observed thatL. acidophilusNCFM could rapidly but transiently upregulate a number of effector genes encoding cytokines and chemokines such as interleukin 1α (IL-1α), IL-1β, CCL2, and CCL20 and that cytokines showed lower expression levels withL. acidophilusNCFM treatment than chemokines. Moreover,L. acidophilusNCFM could activate a pathogen-associated molecular pattern receptor, Toll-like receptor 2 (TLR2), in intestinal epithelial cell lines. The phosphorylation of NF-κB p65 and p38 mitogen-activated protein kinase (MAPK) in intestinal epithelial cell lines was also enhanced byL. acidophilusNCFM. Furthermore, inhibitors of NF-κB (pyrrolidine dithiocarbamate [PDTC]) and p38 MAPK (SB203580) significantly reduced cytokine and chemokine production in the intestinal epithelial cell lines stimulated byL. acidophilusNCFM, suggesting that both NF-κB and p38 MAPK signaling pathways were important for the production of cytokines and chemokines induced byL. acidophilusNCFM.

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Ca2+-sensing receptors in intestinal epithelium

AJP Cell Physiology ◽

10.1152/ajpcell.1997.273.4.c1168 ◽

1997 ◽

Vol 273 (4) ◽

pp. C1168-C1175 ◽

Cited By ~ 161

Author(s):

Lucio Gama ◽

Lynn M. Baxendale-Cox ◽

Gerda E. Breitwieser

Keyword(s):

Epithelial Cell ◽

Epithelial Cells ◽

Intestinal Epithelium ◽

Intestinal Epithelial Cell ◽

Intestinal Epithelial Cells ◽

Northern Blotting ◽

Intestinal Epithelial ◽

Pcr Products ◽

Intracellular Ca ◽

Ht 29

Expression of Ca2+-sensing receptors (CaR) was demonstrated in several human intestinal epithelial cell lines (T84, HT-29, and Caco-2) and in rat intestinal epithelium by both reverse transcriptase-polymerase chain reaction (PCR) and Northern blotting of RNA. Restriction patterns of the PCR products were of the sizes predicted by the human and rat sequences. CaR agonists (Ca2+, poly-l-arginine, protamine) mediated an increase in intracellular Ca2+ in HT-29–18-C1 cells (monitored by changes in fura 2 fluorescence), which was dependent on release from thapsigargin-sensitive stores. U-73122, an inhibitor of phosphatidylinositol-phospholipase C, eliminated the CaR agonist-mediated rise in intracellular Ca2+, whereas its inactive analog, U-73343, had no effect. Pertussis toxin pretreatment had no effect on CaR agonist-mediated modulation of intracellular Ca2+. Taken together, these studies demonstrate that CaR are expressed in intestinal epithelial cells and couple to mobilization of intracellular Ca2+. The presence of CaR in intestinal epithelial cells presents a new locus for investigations into the role(s) of extracellular Ca2+ in modulating intestinal epithelial cell differentiation and transepithelial Ca2+ transport.

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ADAM-15 inhibits wound healing in human intestinal epithelial cell monolayers

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00262.2004 ◽

2005 ◽

Vol 288 (2) ◽

pp. G346-G353 ◽

Cited By ~ 35

Author(s):

Laetitia Charrier ◽

Yutao Yan ◽

Adel Driss ◽

Christian L. Laboisse ◽

Shanthi V. Sitaraman ◽

...

Keyword(s):

Wound Healing ◽

Epithelial Cell ◽

Epithelial Cells ◽

Intestinal Epithelial Cell ◽

Intestinal Epithelial Cells ◽

Epithelial Cell Line ◽

Intestinal Epithelial ◽

Coding Region ◽

Human Intestinal Epithelial Cell ◽

New Variant

The disintegrin metalloproteases (or ADAMs) are membrane-anchored glycoproteins that have been implicated in cell-cell or cell-matrix interactions and in proteolysis of molecules on the cell surface. The expression and/or the pathophysiological implications of ADAMs are not known in intestinal epithelial cells. Therefore, our aim was to investigate the expression and the role of ADAMs in intestinal epithelial cells. Expression of ADAMs was assessed by RT-PCR, Western blot analysis, and immunufluorescence experiments. Wound-healing experiments were performed by using the electric cell substrate impedence sensing technology. Our results showed that ADAMs-10, -12, and -15 mRNA are expressed in the colonic human cell lines Caco2-BBE and HT29-Cl.19A. An ADAM-15 complementary DNA cloned from Caco2-BBE poly(A)+ RNA, and encompassing the entire coding region, was found to be shorter and to present a different region encoding the cytoplasmic tail compared with ADAM-15 sequence deposited in the database. In Caco2-BBE cells and colonic epithelial cells, ADAM-15 protein was found in the apical, basolateral, and intracellular compartments. We also showed that the overexpression of ADAM-15 reduced cell migration in a wound-healing assay in Caco2-BBE monolayers. Our data show that 1) ADAM-15 is expressed in human intestinal epithelia, 2) a new variant of ADAM-15 is expressed in a human intestinal epithelial cell line, and 3) ADAM-15 is involved in intestinal epithelial cells wound-healing processes. Together, these results suggest that ADAM-15 may have important pathophysiological roles in intestinal cells.

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62 Evaluating detoxification efficacy of deoxynivalenol by encapsulated sodium metabisulfite (SMBS) using an in vitro intestinal epithelial cell IPEC-J2 model

Journal of Animal Science ◽

10.1093/jas/skaa278.069 ◽

2020 ◽

Vol 98 (Supplement_4) ◽

pp. 38-39

Author(s):

Peng Lu ◽

Changning Yu ◽

Shangxi Liu ◽

Joshua Gong ◽

Song Liu ◽

...

Keyword(s):

Small Intestine ◽

Epithelial Cell ◽

Epithelial Cells ◽

Intestinal Epithelial Cell ◽

Intestinal Epithelial Cells ◽

In Vitro Release ◽

Simulated Gastric Fluid ◽

Sodium Metabisulfite ◽

Intestinal Epithelial

Abstract Deoxynivalenol (DON) contamination occurs on feed ingredients and causes a reduction in growth performance, damage to the intestinal epithelial cells, and increased susceptibility to enteric pathogen challenge. Sodium metabisulfite (SMBS) has been successfully used to destroy DON in processed grains or feeds. However, SMBS degrades quickly under aqueous acid conditions, such as pig stomachs, and when SMBS is added to diet, little will remain intact in the small intestine where an optimal pH environment exists for detoxification by SMBS. Thus, this study was to encapsulate SMBS into microparticles to deliver intact SMBS to the small intestine and evaluate its efficacy of DON detoxification in the simulated intestine fluid (SIF) using an in vitro intestinal epithelial cell (IPEC-J2) model. The results showed that around 40% of the SMBS loading capacity was achieved in the microparticles. In vitro release studies showed that 1.61% of encapsulated SMBS was released in the simulated gastric fluid (SGF), and the majority of encapsulated SMBS (75.52%) was progressively released in the SIF within 6 h at 37 °C. In vitro cell experiments showed that DON treated with the SIF containing 0.5% SMBS for 2 h completely attenuated the DON-induced cytotoxicity. When DON was treated with the SGF containing 0.5% encapsulated SMBS for 2 h and then the mixture was mixed with the SIF (1:1) and incubated for 2 h, it also completely attenuated the DON-induced cytotoxicity. Moreover, DON treated with the simulated fluid containing 0.5% encapsulated SMBS completely attenuated the gene expression inflammatory cytokines upregulated by DON and restored trans-epithelial electrical resistance (TEER) and tight junction and cytoskeleton. In summary, the encapsulation of SMBS was stable in SGF and allowed a progressive release of SMBS in the SIF. Moreover, the released SMBS in the SIF effectively attenuated the adverse effects induced by DON in the intestinal epithelial cells.

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Factors affecting the colonization of isolated rabbit intestinal epithelial cells by Vibrio cholerae 01 in vitro

Canadian Journal of Microbiology ◽

10.1139/m89-103 ◽

1989 ◽

Vol 35 (6) ◽

pp. 642-645 ◽

Cited By ~ 3

Author(s):

D. Sasmal ◽

B. Guhathakurta ◽

S. N. Sikdar ◽

A. Datta

Keyword(s):

Epithelial Cell ◽

Epithelial Cells ◽

Vibrio Cholerae ◽

Intestinal Epithelial Cell ◽

Intestinal Epithelial Cells ◽

Growth Media ◽

Intestinal Epithelial ◽

Factors Affecting ◽

O Antigen

The adhesive capability of Vibrio cholerae 01 strains to isolated rabbit intestinal epithelial cells was maximally expressed when the bacteria were grown in synthetic broth and was enhanced by the presence of Ca2+ in the growth media. N-Acetyl-D-glucosamine could inhibit the adhesion of the bacteria to rabbit intestinal epithelial cells as could lipopolysaccharide O-antigen from Vibrio cholerae 01 and lectin from Triticum vulgaris. Since the lipopolysaccharide is known to contain N-acetyl-D-glucosamine and because the lectin from Triticum vulgaris shows specificity for this sugar, it is probable that N-acetyl-D-glucosamine is actively involved in the adhesion of Vibrio cholerae 01 to isolated rabbit intestinal epithelial cells.Key words: adhesion, Vibrio cholerae 01, rabbit intestinal epithelial cell.

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