scholarly journals Development of an endogenous epithelial Na+/H+exchanger (NHE3) in three clones of Caco-2 cells

1999 ◽  
Vol 277 (2) ◽  
pp. G292-G305 ◽  
Author(s):  
Andrzej J. Janecki ◽  
Marshall H. Montrose ◽  
C. Ming Tse ◽  
Fermin Sanchez de Medina ◽  
Alain Zweibaum ◽  
...  
Keyword(s):  
Membrane Domains ◽  
Intestinal Epithelial ◽  
Major Mechanism ◽  
Kinase C ◽  
Cell Clones ◽  
Nhe1 Activity ◽  
Epidermal Growth ◽  
Small Intestinal ◽  
Vitro Model

Expression of endogenous Na+/H+exchangers (NHEs) NHE3 and NHE1 at the apical (AP) and basolateral (BL) membrane domains was investigated in three clones (ATCC, PF-11, and TC-7) derived from the human adenocarcinoma cell line Caco-2. In all three clones, NHE1 was the only isoform detected at the BL domain during 3 to 22 postconfluent days (PCD). In clone PF-11, the BL NHE1 activity increased up to 7 PCD and remained stable thereafter. Both NHE1 and NHE3 were found at the AP domain at 3 PCD and contributed 67 and 33% to the total AP Na+/H+exchange, respectively. The AP NHE3 activity increased significantly from 3 to 22 PCD, from 93 to 450 μM H+/s, whereas AP NHE1 activity decreased from 192 to 18 μM H+/s during that time. Similar results were obtained with the ATCC clone, whereas very little AP NHE3 activity was observed in clone TC-7. Surface biotinylation and indirect immunofluorescence confirmed these results and also suggested an increase in the number of cells expressing NHE3 being the major mechanism of the observed overall increase in NHE3 activity in PF-11 and ATCC clones. Phorbol 12-myristate 13-acetate (PMA, 1 μM) acutely inhibited NHE3 activity by 28% of control, whereas epidermal growth factor (EGF, 200 ng/ml) stimulated the activity by 18%. The effect of PMA was abolished by the protein kinase C (PKC) inhibitor 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine, suggesting involvement of PKC in the PMA-induced inhibition of NHE3. Similar magnitude of inhibition by PMA and stimulation by EGF was observed at 7 and 17 PCD, suggesting the development of regulatory mechanisms in the early postconfluent period. Taken together, these data suggest a close similarity of membrane targeting and regulation of endogenous NHE3 between Caco-2 cells and native small intestinal epithelial cells and support the usefulness of some Caco-2 cell clones as an in vitro model for studies on physiology of NHE3 in the intestinal epithelium.

Epidermal growth factor regulation of rat NHE2 gene expression

2001 ◽  
Vol 281 (2) ◽  
pp. C504-C513 ◽  
Author(s):  
Hua Xu ◽  
James F. Collins ◽  
Liqun Bai ◽  
Pawel R. Kiela ◽  
Ronald M. Lynch ◽  
...  
Keyword(s):  
Gene Expression ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Membrane Vesicle ◽  
Mrna Abundance ◽  
Intestinal Epithelial ◽  
Intestinal Brush Border Membrane ◽  
Epidermal Growth ◽  
Vitro Model

Epidermal growth factor (EGF) is involved in acute regulation of Na+/H+ exchangers (NHEs), but the effect of chronic EGF administration on NHE gene expression is unknown. The present studies showed that EGF treatment increased NHE2-mediated intestinal brush-border membrane vesicle Na+ absorption and NHE2 mRNA abundance by nearly twofold in 19-day-old rats. However, no changes were observed in renal NHE2 mRNA or intestinal and renal NHE3 mRNA abundance. To understand the mechanism of this regulation, we developed the rat intestinal epithelial (RIE) cell as an in vitro model to study the effect of EGF on NHE2 gene expression. EGF increased functional NHE2 activity and mRNA abundance in cultured RIE cells, and this stimulation could be blocked by actinomycin D (a transcriptional inhibitor). Additionally, NHE2 promoter reporter gene assays in transiently transfected RIE cells showed an almost twofold increase in promoter activity after EGF treatment. We conclude that rat NHE2 activity can be stimulated by chronic EGF treatment and that this response is at least partially mediated by gene transcription.

scholarly journals Epidermal Growth Factor Inhibits Campylobacter jejuni-Induced Claudin-4 Disruption, Loss of Epithelial Barrier Function, and Escherichia coli Translocation

2008 ◽  
Vol 76 (8) ◽  
pp. 3390-3398 ◽  
Author(s):  
Jennifer M. Lamb-Rosteski ◽  
Lisa D. Kalischuk ◽  
G. Douglas Inglis ◽  
Andre G. Buret
Keyword(s):  
Escherichia Coli ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Campylobacter Jejuni ◽  
Egf Receptor ◽  
Epithelial Barrier ◽  
Intestinal Epithelial ◽  
Epithelial Barrier Function ◽  
Epidermal Growth

ABSTRACT Campylobacter jejuni is a leading cause of acute bacterial enteritis in humans. Poultry serves as a major reservoir of C. jejuni and is thought to act as a principal vehicle of transmission to humans. Epidermal growth factor (EGF) is a small amino acid peptide that exerts a broad range of activities on the intestinal epithelium. The aims of this study were to determine the effect of EGF on C. jejuni intestinal colonization in newly hatched chicks and to characterize its effects on C. jejuni-induced intestinal epithelial barrier disruption. White Leghorn chicks were treated with EGF daily, starting 1 day prior to C. jejuni infection, and were compared to control and C. jejuni-infected, EGF-treated chicks. Infected chicks shed C. jejuni in their feces throughout the study period. C. jejuni colonized the small intestine and cecum, disseminated to extraintestinal organs, and caused jejunal villus atrophy. EGF reduced jejunal colonization and dissemination of C. jejuni to the liver and spleen. In EGF-treated C. jejuni-infected chicks, villus height was not significantly different from that in untreated C. jejuni-infected chicks or controls. In vitro, C. jejuni attached to and invaded intestinal epithelial cells, disrupted tight junctional claudin-4, and increased transepithelial permeability. C. jejuni also promoted the translocation of noninvasive Escherichia coli C25. These C. jejuni-induced epithelial abnormalities were abolished by pretreatment with EGF, and the effect was dependent upon activation of the EGF receptor. These findings highlight EGF's ability to alter colonization of C. jejuni in the intestinal tract and to protect against pathogen-induced barrier defects.

scholarly journals In vitro assessment of the starch digestibility of western Canadian wheat market classes and cultivars

2018 ◽  
Vol 98 (3) ◽  
pp. 463-476 ◽  
Author(s):  
Namalika D. Karunaratne ◽  
Dawn A. Abbott ◽  
Ravindra N. Chibbar ◽  
Pierre J. Hucl ◽  
Curtis J. Pozniak ◽  
...  
Keyword(s):  
Crude Protein ◽  
In Vitro Model ◽  
Starch Digestibility ◽  
Wheat Market ◽  
In Vitro Assessment ◽  
Market Class ◽  
Small Intestinal ◽  
Vitro Model ◽  
Total Starch

The objective of the study was to measure the effect of wheat market class and cultivar on starch digestibility using an in vitro model that mimics the chicken digestive tract and relate it to grain characteristics. The study evaluated 18 wheat cultivars from eight western Canadian wheat classes and, each cultivar was replicated four times. Samples were subjected to gastric and small intestine (SI) digestion phases and each sample was assayed in triplicate; glucose release was measured in SI phase. Starch granule distribution, amylose, total starch, crude protein (CP), ash, and non-starch polysaccharides (NSP) were analyzed in all wheat samples. Small intestinal phase times of 15, 60, and 120 min were chosen to approximate digestion in the terminal duodenum, jejunum, and ileum. Starch digestibility of wheat classes ranged as follows: 15 min — 33.1% to 49.1%, 60 min — 80.2% to 93.3%, and 120 min — 92.4% to 97.6%. Starch digestibility positively correlated with CP, ash, NSP, and proportion of large granules, whereas it negatively correlated with total starch, and proportion of small and medium granules. In conclusion, market class and cultivar of western Canadian wheat affects both rate and extent of starch digestibility and it is related to various grain characteristics.

Interleukin-1β enhances retinoic acid-mediated expression of bone-type alkaline phosphatase in rat IEC-6 cells

2001 ◽  
Vol 280 (3) ◽  
pp. G510-G517 ◽  
Author(s):  
Takeshi Nikawa ◽  
Madoka Ikemoto ◽  
Kaori Tokuoka ◽  
Shigetada Teshima ◽  
David H. Alpers ◽  
...  
Keyword(s):  
Alkaline Phosphatase ◽  
Retinoic Acid ◽  
Retinoic Acid Receptor ◽  
Transcript Level ◽  
Intestinal Epithelial ◽  
Alp Activity ◽  
Kinase C ◽  
Fetal Rat ◽  
Epidermal Growth

We previously showed that vitamin A upregulated the expression of bone-type alkaline phosphatase (ALP) in fetal rat small intestine and rat intestinal IEC-6 cells. In this study, we examined interactions between retinoic acid (RA) and several growth factors/cytokines on the isozyme expression in IEC-6 cells. Epidermal growth factor and interleukins (ILs)-2, -4, -5, and -6 completely blocked the RA-mediated increase in ALP activity. In contrast, IL-1β markedly increased the activity, protein, and mRNA of the bone-type ALP only when RA was present. IL-1β and/or RA did not change the type 1 IL-1 receptor transcript level, whereas IL-1β enhanced the RA-induced expressions of retinoic acid receptor-β (RAR-β) and retinoid X receptor-β (RXR-β) mRNAs and RA-mediated RXR response element binding. The synergism of IL-1β and RA on ALP activity was completely blocked by protein kinase C (PKC) inhibitors. Our results suggest that IL-1β may modify the ALP isozyme expression in small intestinal epithelial cells by stimulating PKC-dependent, RAR-β- and/or RXR-β-mediated signaling pathways.

scholarly journals Protein Kinase C Signaling Mediates a Program of Cell Cycle Withdrawal in the Intestinal Epithelium

2000 ◽  
Vol 151 (4) ◽  
pp. 763-778 ◽  
Author(s):  
Mark R. Frey ◽  
Jennifer A. Clark ◽  
Olga Leontieva ◽  
Joshua M. Uronis ◽  
Adrian R. Black ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Intestinal Epithelium ◽  
Molecular Mechanisms ◽  
Model Systems ◽  
Intestinal Epithelial ◽  
Kinase C

Members of the protein kinase C (PKC) family of signal transduction molecules have been widely implicated in regulation of cell growth and differentiation, although the underlying molecular mechanisms involved remain poorly defined. Using combined in vitro and in vivo intestinal epithelial model systems, we demonstrate that PKC signaling can trigger a coordinated program of molecular events leading to cell cycle withdrawal into G0. PKC activation in the IEC-18 intestinal crypt cell line resulted in rapid downregulation of D-type cyclins and differential induction of p21waf1/cip1 and p27kip1, thus targeting all of the major G1/S cyclin-dependent kinase complexes. These events were associated with coordinated alterations in expression and phosphorylation of the pocket proteins p107, pRb, and p130 that drive cells to exit the cell cycle into G0 as indicated by concomitant downregulation of the DNA licensing factor cdc6. Manipulation of PKC isozyme levels in IEC-18 cells demonstrated that PKCα alone can trigger hallmark events of cell cycle withdrawal in intestinal epithelial cells. Notably, analysis of the developmental control of cell cycle regulatory molecules along the crypt–villus axis revealed that PKCα activation is appropriately positioned within intestinal crypts to trigger this program of cell cycle exit–specific events in situ. Together, these data point to PKCα as a key regulator of cell cycle withdrawal in the intestinal epithelium.

scholarly journals Copper Transport and Disease: What Can We Learn from Organoids?

2019 ◽  
Vol 39 (1) ◽  
pp. 75-94 ◽  
Author(s):  
Hannah Pierson ◽  
Haojun Yang ◽  
Svetlana Lutsenko
Keyword(s):  
Three Dimensional ◽  
Transport Processes ◽  
Intestinal Epithelial ◽  
Metabolic Coupling ◽  
Significant Research ◽  
Cu Homeostasis ◽  
Vitro Model ◽  
Cu Uptake ◽  
Fat Processing

Many metals have biological functions and play important roles in human health. Copper (Cu) is an essential metal that supports normal cellular physiology. Significant research efforts have focused on identifying the molecules and pathways involved in dietary Cu uptake in the digestive tract. The lack of an adequate in vitro model for assessing Cu transport processes in the gut has led to contradictory data and gaps in our understanding of the mechanisms involved in dietary Cu acquisition. The recent development of organoid technology has provided a tractable model system for assessing the detailed mechanistic processes involved in Cu utilization and transport in the context of nutrition. Enteroid (intestinal epithelial organoid)-based studies have identified new links between intestinal Cu metabolism and dietary fat processing. Evidence for a metabolic coupling between the dietary uptake of Cu and uptake of fat (which were previously thought to be independent) is a new and exciting finding that highlights the utility of these three-dimensional primary culture systems. This review has three goals: ( a) to critically discuss the roles of key Cu transport enzymes in dietary Cu uptake; ( b) to assess the use, utility, and limitations of organoid technology in research into nutritional Cu transport and Cu-based diseases; and ( c) to highlight emerging connections between nutritional Cu homeostasis and fat metabolism.

Differential expression and regulation of ADAM17 and TIMP3 in acute inflamed intestinal epithelia

2009 ◽  
Vol 296 (6) ◽  
pp. G1332-G1343 ◽  
Author(s):  
Annabelle Cesaro ◽  
Abakar Abakar-Mahamat ◽  
Patrick Brest ◽  
Sandra Lassalle ◽  
Eric Selva ◽  
...  
Keyword(s):  
High Activity ◽  
Early Phase ◽  
Active Form ◽  
Tissue Inhibitor Of Metalloproteinase ◽  
Intestinal Epithelial ◽  
T84 Cells ◽  
Intestinal Epithelia ◽  
Tnf Α ◽  
Vitro Model

The acute phase of Crohn's disease (CD) is characterized by a large afflux of polymorphonuclear leukocytes (PMNL) into the mucosa and by the release of TNF-α. Conversion of inactive TNF-α into an active form requires the cleavage of a transmembrane TNF-α precursor by the TNF-α-converting enzyme (ADAM17), a protease mainly regulated by the tissue inhibitor of metalloproteinase 3 (TIMP3). The aim of the present study was to investigate in an in vitro model of PMNL transepithelial migration and in the intestinal mucosa of patients with CD the expression and regulation of ADAM17 and TIMP3 in intestinal epithelial cells (IEC). ADAM17 and TIMP3 expression was analyzed by Western blotting, RT-PCR, confocal microscopy, and immunohistochemistry by using the T84 model and digestive biopsies. ADAM17 expression in IEC was increased at a posttranscriptional level during the early phase (from 2 to 4 h) of PMNL transepithelial migration whereas TIMP3 was only increased 24 h later. TNF-α induced an early upregulation of ADAM17 in T84 cells, whereas PMNL adhesion, H2O2, or epithelial tight junction opening alone did not affect the amount of ADAM17. Immunohistochemistry of intestinal biopsies revealed that strong expression of ADAM17 was associated with a high activity of CD. In contrast, TIMP3 was very poorly expressed in these biopsies. ADAM17 and TIMP3 profiling did not correlated with the NOD2/CARD15 status. The ADAM17 activity was higher both in the early phase of PMNL transepithelial migration and in active CD. These results showed early posttranscriptional upregulation of ADAM17 in IEC linked to PMNL transepithelial migration and a high activity of CD.

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