Increased abundance of cytoplasmic and nuclear caveolin 1 in human diploid fibroblasts in H2O2-induced premature senescence and interplay with p38αMAPK

AbstractTreatment of IMR-90 human diploid fibroblasts with a sublethal concentration of H2O2 induces premature senescence. We investigated the protein abundance, subcellular localization and involvement of caveolin 1 in premature senescence. Caveolin 1 is a scaffolding protein able to concentrate and organize signaling molecules within the caveolae membrane domains. We report the first evidence of increased nuclear and cytoplasmic localization of caveolin 1 during establishment of H2O2-induced premature senescence. Moreover, we demonstrate that phosphorylation of caveolin 1 during treatment with H2O2 is dependent on p38α mitogen-activated protein kinase

Anti-inflammatory effects of dietary phenolic compounds in an in vitro model of inflamed human intestinal epithelium

Phenolic compounds (PCs) are considered to possess anti-inflammatory properties and therefore were proposed as an alternative natural approach to prevent or treat chronic inflammatory diseases. However their effects are not fully understood, particularly at the intestinal level. To further understand their mode of action at the molecular level during intestinal inflammation, an in vitro model of inflamed human intestinal epithelium was established. Different representative dietary PCs, i.e. resveratrol, ellagic and ferulic acids, curcumin, quercetin, chrysin, (−)-epigallocatechin-3-gallate (EGCG) and genistein, were selected. To mimic intestinal inflammation, differentiated Caco-2 cells cultivated in bicameral inserts, in a serum-free medium, were treated with a cocktail of pro-inflammatory substances: interleukin (IL)-1β, tumor necrosis factor-α, interferon-γ and lipopolysaccharides. The inflammatory state was characterized by a leaky epithelial barrier (attenuation of the transepithelial electrical resistance) and by an over-expression at the mRNA and protein levels for pro-inflammatory markers, i.e. IL-6, IL-8 and monocyte chemoattractant protein-1 (MCP-1), quantified by ELISA and by gene expression analysis using a low-density array allowing the evaluation of expression level for 46 genes relevant of the intestinal inflammation and functional metabolism. Treatment with PCs, used at a realistic intestinal concentration, did not affect cell permeability. In inflamed cells, the incubation with genistein reduced the IL-6 and MCP-1 overproduction, to ca. 50% of the control, whereas EGCG provoked a decrease in the IL-6 and IL-8 over-secretion, by 50 and 60%, respectively. This occurred for both flavonoids without any concomitant inhibition of the corresponding mRNA expression. All the PCs generated a specific gene expression profile, with genistein the most efficient in the downregulation of the expression, or over-expression, of inflammatory genes notably those linked to the arachidonic metabolism pathway. In conclusion, this study provides evidence that genistein and EGCG downregulate the inflammatory response in inflamed intestinal epithelial cells by a pathway implicating largely a post-transcriptional regulatory mechanism

Dimethyl sulfoxide (DMSO) attenuates the inflammatory response in the in vitro intestinal Caco-2 cell model.

This study aimed to investigate dose effects of dimethyl sulfoxide (DMSO) (0.05-1%) on the intestinal inflammatory response in confluent- and differentiated-Caco-2 cells stimulated with interleukin (IL)-1β or a pro-inflammatory cocktail for 24h. Cyclooxygenase-2 (COX-2) activity was assayed by incubating inflamed cells with arachidonic acid and then measuring prostaglandin-E(2) (PGE(2)) produced. Soluble mediators (IL-8, IL-6, macrophage chemoattractant protein-1 (MCP-1), and COX-2-derived PGE(2)) were quantified by enzyme immunoassays and mRNA expression of 33 proteins by high throughput TaqMan Low Density Array. Data showed that DMSO decreased induced IL-6 and MCP-1 secretions in a dose-dependent manner (P<0.05), but not IL-8; these effects were cell development- and stimulus- independent. Moreover, in IL-1β-stimulated confluent-cells, DMSO dose-dependently reduced COX-2-derived PGE(2) (P<0.05). DMSO at 0.5% decreased significantly mRNA levels of 14 proteins involved in the inflammatory response (including IL-6, IL-1α, IL-1β, and COX-2). Thus, DMSO at low concentrations (0.1-0.5%) exhibits anti-inflammatory properties in the in vitro intestinal Caco-2 cell model. This point is important to be taken into account when assessing anti-inflammatory properties of bioactive compounds requiring DMSO as vehicle, such as phenolic compounds, in order to avoid miss-interpretation of the results

Deoxynivalenol affects in vitro intestinal epithelial cell barrier integrity through inhibition of protein synthesis

Deoxynivalenol (DON), one of the most common mycotoxin contaminants of raw and processed cereal food, adversely affects the gastrointestinal tract. Since DON acts as a protein synthesis inhibitor, the constantly renewing intestinal epithelium could be particularly sensitive to DON. We analyzed the toxicological effects of DON on intestinal epithelial protein synthesis and barrier integrity. Differentiated Caco-2 cells, as a widely used model of the human intestinal barrier, were exposed to realistic intestinal concentrations of DON (50, 500 and 5000 ng/ml) during 24 h. DON caused a concentration-dependent decrease in total protein content associated with a reduction in the incorporation of [H-3]-leucine, demonstrating its inhibitory effect on protein synthesis. DON simultaneously increased the paracellular permeability of the monolayer as reflected through a decreased transepithelial electrical resistance associated with an increased paracellular flux of the tracer [H-3]-mannitol. A concentration-dependent reduction in the expression level of the tight junction constituent claudin-4 was demonstrated by Western blot, which was not due to diminished transcription, increased degradation, or NF-kappa B, ERK or JNK activation, and was also observed for a tight junction independent protein, i.e. intestinal alkaline phosphatase. These results demonstrate a dual toxicological effect of DON on differentiated Caco-2 cells consisting in an inhibition of protein synthesis as well as an increase in monolayer permeability, and moreover suggest a possible link between them through diminished synthesis of the tight junction constituent claudin-4. (C) 2010 Elsevier Inc. All rights reserved

Physio-pathological parameters affect the activation of inflammatory pathways by deoxynivalenol in Caco-2 cells

The intake of deoxynivalenol (DON), a mycotoxin contaminating cereal food items, causes gastro-intestinal illness in human and animal. This study investigated whether intracellular inflammatory cascades (MAPKs and NF-kappa B), cell maturity (proliferating vs. differentiated), cell state (control vs. inflamed) and exposure duration (chronic vs. acute) affect IL-8 secretion and PGE-2 synthesis in Caco-2 cells exposed to plausible intestinal concentrations (50, 500 and 5000 ng/ml) of DON. IL-8 secretion and PGE-2 synthesizing capacity were dose-dependently upregulated in differentiated Caco-2 cells exposed to DON during 24 h, reaching an increase of similar to 25 and 1.7-fold respectively, whereas transcript level of IL-8 and COX-2 were increased by similar to 40 and 17-fold. Similar results were obtained with proliferating cells. The upregulation decreased upon simultaneous incubation with inhibitors of MAPKs ERK1/2 or p38 or of transcription factor NF-kappa B. IL-8 secretion and PGE-2 synthesizing capacity increased respectively by similar to 15 and 2-fold after chronic 21 day incubation with DON (50 ng/ml). IL-8 production was exacerbated (similar to 510-fold versus negative control) upon simultaneous exposure to inflammatory stimuli. These results suggest activation of inflammatory pathways in intestinal epithelial cells exposed chronically or acutely to DON. The sensitivity to DON, whereas not affected by cell differentiation, is exacerbated by the presence of additional stimuli. (C) 2010 Elsevier Ltd. All rights reserved