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.

scholarly journals Tight Junction Proteins Claudin-2 and -12 Are Critical for Vitamin D-dependent Ca2+ Absorption between Enterocytes

2008 ◽  
Vol 19 (5) ◽  
pp. 1912-1921 ◽  
Author(s):  
Hiroki Fujita ◽  
Kotaro Sugimoto ◽  
Shuichiro Inatomi ◽  
Toshihiro Maeda ◽  
Makoto Osanai ◽  
...  
Keyword(s):  
Vitamin D ◽  
Tight Junction ◽  
Active Form ◽  
Intestinal Epithelial ◽  
Tight Junction Proteins ◽  
Dihydroxyvitamin D ◽  
Intestinal Epithelia ◽  
Junction Proteins

Ca2+ is absorbed across intestinal epithelial monolayers via transcellular and paracellular pathways, and an active form of vitamin D3, 1α,25-dihydroxyvitamin D3 [1α,25(OH)2D3], is known to promote intestinal Ca2+ absorption. However, the molecules driving the paracellular Ca2+ absorption and its vitamin D dependency remain obscure. Because the tight junction proteins claudins are suggested to form paracellular channels for selective ions between neighboring cells, we hypothesized that specific intestinal claudins might facilitate paracellular Ca2+ transport and that expression of these claudins could be induced by 1α,25(OH)2D3. Herein, we show, by using RNA interference and overexpression strategies, that claudin-2 and claudin-12 contribute to Ca2+ absorption in intestinal epithelial cells. We also provide evidence showing that expression of claudins-2 and -12 is up-regulated in enterocytes in vitro and in vivo by 1α,25(OH)2D3 through the vitamin D receptor. These findings strongly suggest that claudin-2- and/or claudin-12-based tight junctions form paracellular Ca2+ channels in intestinal epithelia, and they highlight a novel mechanism behind vitamin D-dependent calcium homeostasis.

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.

Dynamics of endogenous ATP7A (Menkes protein) in intestinal epithelial cells: copper-dependent redistribution between two intracellular sites

2007 ◽  
Vol 292 (4) ◽  
pp. G1181-G1194 ◽  
Author(s):  
L. Nyasae ◽  
R. Bustos ◽  
L. Braiterman ◽  
B. Eipper ◽  
A. Hubbard
Keyword(s):  
Cell Surface ◽  
Basolateral Membrane ◽  
Cell Periphery ◽  
Intestinal Epithelial ◽  
Intestinal Epithelia ◽  
Dependent Behavior ◽  
Cell Surface Biotinylation ◽  
Vitro Model

We report for the first time on the copper-dependent behavior of endogenous ATP7A in two types of polarized intestinal epithelia, rat enterocytes in vivo and filter-grown Caco-2 cells, an accepted in vitro model of human small intestine. We used high-resolution, confocal immunofluorescence combined with quantitative cell surface biotinylation and found that the vast majority of endogenous ATP7A was localized intracellularly under all copper conditions. In copper-depleted cells, virtually all of the ATP7A localized to a post-TGN compartment, with <3% of the total protein detectable at the basolateral cell surface. When copper levels were elevated, ATP7A dispersed to the cell periphery in punctae whose pattern did not overlap with the steady-state distributions of post-Golgi, endosomal, or basolateral membrane markers; only ∼8–10% of the recovered ATP7A was detected at the basolateral cell surface. These results raise several questions regarding prevailing models of ATP7A dynamics and the mechanism of copper efflux.

scholarly journals Antioxidant and Hypolipidemic Activity of Açai Fruit Makes It a Valuable Functional Food

Antioxidants ◽  
2020 ◽  
Vol 10 (1) ◽  
pp. 40
Author(s):  
Anna Virginia Adriana Pirozzi ◽  
Paola Imbimbo ◽  
Antonella D’Agostino ◽  
Virginia Tirino ◽  
Rosario Finamore ◽  
...  
Keyword(s):  
Functional Food ◽  
Subcutaneous Fat ◽  
Cell Models ◽  
Alcoholic Fatty Liver ◽  
Hypoxic Conditions ◽  
Tnf Α ◽  
Different Types ◽  
Vitro Model ◽  
Alcoholic Fatty Liver Disease

Several plant extracts are acquiring increasing value because of their antioxidant activity and hypolipidemic properties. Among them, great interest has been recently paid to açai fruit as a functional food. The aim of this study was to test the ability of açai extract in reducing oxidative stress and modulating lipid metabolism in vitro using different cell models and different types of stress. In fact, lipid peroxidation as evaluated in a HepG2 model was reduced five-fold when using 0.25 µg/mL of extract, and it was further reduced (20-fold) with the concentration increase up to 2.5 µg/mL. With the non alcoholic fatty liver disease (NAFLD)in vitro model, all concentrations tested showed at least a two-fold reduced fat deposit. In addition, primary adipocytes challenged with TNF-α under hypoxic conditions to mimic the persistent subcutaneous fat, treated with açai extract showed an approximately 40% reduction of fat deposit. Overall, our results show that açai is able to counteract oxidative states in all the cell models analysed and to prevent the accumulation of lipid droplets. No toxic effects and high stability overtime were highlighted at the concentrations tested. Therefore, açai can be considered a suitable support in the prevention of different alterations of lipid and oxidative metabolism responsible for fat deposition and metabolic pathological conditions.

Functional coupling of tight junctions and microfilaments in T84 monolayers

1988 ◽  
Vol 254 (3) ◽  
pp. G416-G423 ◽  
Author(s):  
J. L. Madara ◽  
J. Stafford ◽  
D. Barenberg ◽  
S. Carlson
Keyword(s):  
Tight Junction ◽  
Actin Binding ◽  
Functional Coupling ◽  
Flux Analysis ◽  
Intestinal Epithelial ◽  
Binding Agent ◽  
T84 Cells ◽  
Intestinal Epithelia ◽  
Tight Junction Permeability ◽  
Cd Exposure

The actin-binding agent cytochalasin D (CD) in intact intestinal epithelium appears to elicit segmentation and contraction of a perijunctional ring of actomyosin and, consequently, to diminish tight junction resistance. We determined if an intestinal epithelial model composed of T84 cells also displayed such a perijunctional cytoskeletal specialization and, if so, whether exposure to CD also affected the tight junction barrier. We find T84 cells display a prominent perijunctional microfilament ring that is actin rich. CD elicits large decreases in transepithelial resistance due specifically to perturbed tight junction permeability as determined with dual Na+-mannitol flux analysis. Transepithelial mannitol and insulin fluxes also increase after CD exposure, but these molecules remain differentially restricted in accordance with their sizes, indicating that gross disruption of the monolayer has not occurred. Structurally, CD elicits segmentation and condensation of the perijunctional ring into actin-rich plaques. These features have similarity to those seen in native intestinal epithelia. This system may represent a simple model for studies of cytoskeletal-tight junction relationships.

scholarly journals Effect of the proinflammatory cytokine TNF-α on intestinal riboflavin uptake: inhibition mediated via transcriptional mechanism(s)

2018 ◽  
Vol 315 (5) ◽  
pp. C653-C663 ◽  
Author(s):  
Kasin Yadunandam Anandam ◽  
Omar A. Alwan ◽  
Veedamali S. Subramanian ◽  
Padmanabhan Srinivasan ◽  
Rubina Kapadia ◽  
...  
Keyword(s):  
Ex Vivo ◽  
Significant Inhibition ◽  
Mrna Levels ◽  
In Vivo Models ◽  
Uptake Inhibition ◽  
Tnf Α ◽  
Vitro Model ◽  
Factor Α

Riboflavin (RF), is essential for normal cellular metabolism/function. Intestinal RF absorption occurs via a specific carrier-mediated process that involves the apical transporter RFVT-3 ( SLC52A3) and the basolateral RFVT-1 (SLC52A1). Previously, we characterized different cellular/molecular aspects of the intestinal RF uptake process, but nothing is known about the effect of proinflammatory cytokines on the uptake event. We addressed this issue using in vitro, ex vivo, and in vivo models. First, we determined the level of mRNA expression of the human (h)RFVT-3 and hRFVT-1 in intestinal tissue of patients with inflammatory bowel disease (IBD) and observed a markedly lower level compared with controls. In the in vitro model, exposing Caco-2 cells to tumor necrosis factor-α (TNF-α) led to a significant inhibition in RF uptake, an effect that was abrogated upon knocking down TNF receptor 1 (TNFR1). The inhibition in RF uptake was associated with a significant reduction in the expression of hRFVT-3 and -1 protein and mRNA levels, as well as in the activity of the SLC52A3 and SLC52A1 promoters. The latter effects appear to involve Sp1 and NF-κB sites in these promoters. Similarly, exposure of mouse small intestinal enteroids and wild-type mice to TNF-α led to a significant inhibition in physiological and molecular parameters of intestinal RF uptake. Collectively, these findings demonstrate that exposure of intestinal epithelial cells to TNF-α leads to inhibition in RF uptake and that this effect is mediated, at least in part, via transcriptional mechanism(s). These findings may explain the significantly low RF levels observed in patients with IBD.

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.

scholarly journals A Novel Lactic Acid Bacteria Mixture: Macrophage-Targeted Prophylactic Intervention in Colorectal Cancer Management

2020 ◽  
Vol 8 (3) ◽  
pp. 387
Author(s):  
Petra Hradicka ◽  
Jane Beal ◽  
Monika Kassayova ◽  
Andrew Foey ◽  
Vlasta Demeckova
Keyword(s):  
Colorectal Cancer ◽  
Male Rats ◽  
Cancer Management ◽  
Immune Parameters ◽  
Chemically Induced ◽  
Tnf Α ◽  
Vitro Model ◽  
Macrophage Subsets

Colorectal cancer (CRC) is one of the most common forms of cancer. Its onset from chronic inflammation is widely accepted. Moreover, dysbiosis plays an undeniable role, thus the use of probiotics in CRC has been suggested. They exhibit both anti- and pro-inflammatory properties and restore balance in the microbiota. The aim of this study was to investigate the immunomodulatory properties of six lactobacilli with probiotic features in an in vitro model of macrophage-like cells and to test these pooled probiotics for their anti-tumour properties in a chemically induced CRC model using Wistar male rats. Upon co-culture of M1- and M2-like macrophages with lactobacilli, cytokine release (TNF-α, IL-1β, IL-18, IL-23) and phagocytic activity using fluorescent-labelled bacteria were tested. The effects of orally administered probiotics on basic cancer and immune parameters and cytokine concentration (TNF-α, IL-1β, IL-18) in colon tumours were studied. Tested lactobacilli exhibited both pro- and anti-inflammatory properties in in vitro conditions. In vivo study showed that the administration of probiotics was able to decrease multiplicity, volume and total tumour numbers, restore colon length (p < 0.05) and increase IL-18 production (p < 0.05) in tumour tissue. These data indicate both an immunomodulatory effect of probiotics on distinct macrophage subsets and a protective effect against chemically-induced CRC.

Beneficial effects of nontoxic ozone on H2O2-induced stress and inflammation

2016 ◽  
Vol 94 (6) ◽  
pp. 577-583 ◽  
Author(s):  
Altug Kucukgul ◽  
Suat Erdogan ◽  
Ramazan Gonenci ◽  
Gonca Ozan
Keyword(s):  
Cell Loss ◽  
Alveolar Cells ◽  
Sod Activity ◽  
Beneficial Effects ◽  
Tnf Α ◽  
Ozone Pretreatment ◽  
Anti Oxidant ◽  
Anti Inflammatory ◽  
Vitro Model

In this study, the anti-oxidant and anti-inflammatory efficacy of ozone oxidative preconditioning (OOP) were investigated on hydrogen peroxide (H2O2)-induced human lung alveolar cells. In MTT and trypan blue viability tests, while 100 μmol/L H2O2caused a 17.3% and 21.9% decrease in the number of living cells, respectively, ozone at 20 μmol/L regenerated cell proliferation and prevented 9.6% and 11.0% of cell loss, respectively. In addition, H2O2decreased the transcription levels of catalase (CAT), glutathione peroxidase (GPx), and superoxide dismutase (SOD) 5.43-, 2.89-, and 5.33-fold, respectively, while it increased Bax, NF-κβ, TNF-α, and iNOS expression 1.57-, 1.32-, 1.40-, and 1.41-fold, respectively. Ozone pretreatment, however, increased CAT, GPx, and SOD transcription levels 7.08-, 5.17-, and 6.49-fold and decreased Bax, NF-κβ, TNF-α, and iNOS transcriptions by 1.25-, 0.76-, 3.63-, and 7.91-fold, respectively. Moreover, intracellular glutathione (GSH) level and SOD activity were decreased by 46.2% and 45.0% in the H2O2treatment group, and OOP recovered 58.5% and 20.1% of the decreases caused by H2O2. H2O2also increased nitrite levels 7.84-fold, and OOP reduced this increase by half. Consequently, OOP demonstrated potent anti-oxidant and anti-inflammatory effects on in vitro model of oxidative stress-induced lung injury.

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