Loss of Hepatocyte-Nuclear-Factor-4α Affects Colonic Ion Transport and Causes Chronic Inflammation Resembling Inflammatory Bowel Disease in Mice

BACKGROUND: Hnf4alpha, an epithelial specific transcriptional regulator, is decreased in inflammatory bowel disease and protects against chemically-induced colitis in mice. However, the precise role of this factor in maintaining normal inflammatory homeostasis of the intestine remains unclear. The aim of this study was to evaluate the sole role of epithelial Hnf4alpha in the maintenance of gut inflammatory homeostasis in mice. METHODOLOGY/PRINCIPAL FINDINGS: We show here that specific epithelial deletion of Hnf4alpha in mice causes spontaneous chronic intestinal inflammation leading to focal areas of crypt dropout, increased cytokines and chemokines secretion, immune cell infiltrates and crypt hyperplasia. A gene profiling analysis in diseased Hnf4alpha null colon confirms profound genetic changes in cell death and proliferative behaviour related to cancer. Among the genes involved in the immune protection through epithelial barrier function, we identify the ion transporter claudin-15 to be down-modulated early in the colon of Hnf4alpha mutants. This coincides with a significant decrease of mucosal ion transport but not of barrier permeability in young animals prior to the manifestation of the disease. We confirm that claudin-15 is a direct Hnf4alpha gene target in the intestinal epithelial context and is down-modulated in mouse experimental colitis and inflammatory bowel disease. CONCLUSION: Our results highlight the critical role of Hnf4alpha to maintain intestinal inflammatory homeostasis during mouse adult life and uncover a novel function for Hnf4alpha in the regulation of claudin-15 expression. This establishes Hnf4alpha as a mediator of ion epithelial transport, an important process for the maintenance of gut inflammatory homeostasis

Oxidative Stress and Mitochondrial Functions in the Intestinal Caco-2/15 Cell Line

Although mitochondrial dysfunction and oxidative stress are central mechanisms in various pathological conditions, they have not been extensively studied in the gastrointestinal tract, which is known to be constantly exposed to luminal oxidants from ingested foods. Key among these is the simultaneous consumption of iron salts and ascorbic acid, which can cause oxidative damage to biomolecules.The objective of the present work was to evaluate how iron-ascorbate (FE/ASC)-mediated lipid peroxidation affects mitochondrion functioning in Caco-2/15 cells. Our results show that treatment of Caco-2/15 cells with FE/ASC (0.2 mM/2 mM) (1) increased malondialdehyde levels assessed by HPLC; (2) reduced ATP production noted by luminescence assay; (3) provoked dysregulation of mitochondrial calcium homeostasis as evidenced by confocal fluorescence microscopy; (4) upregulated the protein expression of cytochrome C and apoptotic inducing factor, indicating exaggerated apoptosis; (5) affected mitochondrial respiratory chain complexes I, II, III and IV; (6) elicited mtDNA lesions as illustrated by the raised levels of 8-OHdG; (7) lowered DNA glycosylase, one of the first lines of defense against 8-OHdG mutagenicity; and (8) altered the gene expression and protein mass of mitochondrial transcription factors (mtTFA, mtTFB1, mtTFB2) without any effects on RNA Polymerase. The presence of the powerful antioxidant BHT (50 microM) prevented the occurrence of oxidative stress and most of the mitochondrial abnormalities.Collectively, our findings indicate that acute exposure of Caco-2/15 cells to FE/ASC-catalyzed peroxidation produces harmful effects on mitochondrial functions and DNA integrity, which are abrogated by the powerful exogenous BHT antioxidant. Functional derangements of mitochondria may have implications in oxidative stress-related disorders such as inflammatory bowel diseases

Multidrug Resistance-Associated Protein 2 Expression Is Upregulated by Adenosine 5'-Triphosphate in Colorectal Cancer Cells and Enhances Their Survival to Chemotherapeutic Drugs.

Extracellular adenosine 5'-triphosphate (ATP) is a signaling molecule that induces a plethora of effects ranging from the regulation of cell proliferation to modulation of cancerous cell behavior. In colorectal cancer, ATP was reported to stimulate epithelial cell proliferation and possibly promote resistance to anti-cancer treatments. However, the exact role of this danger-signaling molecule on cancerous intestinal epithelial cells (IECs) in response to chemotherapeutic agents remains unknown. To address how ATP may influence the response of cancerous IECs to chemotherapeutic agents, we used Caco-2 cells, which display enterocyte-like features, to determine the effect of ATP on the expression of multidrug resistance-associated protein 2 (MRP2). Gene and protein expression were determined by quantitative real-time PCR (qRT-PCR) and Western blotting. Resistance to etoposide, cisplatin and doxorubicin was determined by MTT assays in response to ATP stimulation of Caco-2 cells and in cells for which MRP2 expression was down-regulated by shRNA. ATP increased the expression of MRP2 at both the mRNA and protein levels. MRP2 expression involved an ATP-dependent stimulation of the MEK/ERK signaling pathway that was associated with an increase in relative resistance of Caco-2 cells to etoposide. Abolition of MRP2 expression using shRNA significantly reduced the protective effect of MRP2 toward etoposide as well as to cisplatin and doxorubicin. This study describes the mechanism by which ATP may contribute to the chemoresistance of cancerous IECs in colorectal cancer. Given the heterogeneity of colorectal adenocarcinoma responses to anti-cancer drugs, these findings call for further study to understand the role of P2 receptors in cancer drug therapy and to develop novel therapies aimed at regulating P2 receptor activity

MRP2 expression is regulated by MEK/ERK signaling cascade.

<p>Caco-2 cells were pretreated with NFκB (2 μM, Bay, BAY11-7082), MEK1/2 (10 μM, U0, U0126), PI3K (20 μM, LY, LY294003) and p38 (20 μM, SB, SB203580) inhibitors for 30 minutes and stimulated with 100 μM ATP for 6 h. <b>(A)</b> A typical Western blot against MRP2 is displayed from which <b>(B)</b> densitometry analysis showed a significant reduction in MRP2 expression in the presence of U0126, a selective MEK 1/2 inhibitor. Cells pretreated with U0126 led to a 75% reduction in the expression of MRP2 compared to ATP-stimulated cells only (-). Results are presented as the mean ± SEM of three separate experiments performed in duplicate. Statistical significance was determined by an unpaired <i>t</i>-test, where * p < 0.05 vs. non-stimulated (N-S) or ATP-stimulated cells as indicated on figure.</p

The down-regulation of MRP2 expression by shRNA renders Caco-2 cells more sensitive to etoposide.

<p><b>(A)</b> Western blot analysis was used to assess the down-regulation of MRP2 protein expression in the presence of two shRNAs directed against the protein. Down-regulation was achieved by lentiviral infection of Caco-2 cells as previously described [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0136080#pone.0136080.ref031" target="_blank">31</a>]. shRNA directed against MRP2 (sh305 and sh307) abolished protein expression by 90–100% comparatively to cells expressing a non-targeting shRNA (shNT). <b>(B)</b> Caco-2 cells stably expressing shNT or shMRP2 (#305) were incubated with the cytotoxic drug etoposide for 84 h. Sensitivity to the anti-cancer drug was determined by the MTT cell viability assay. A dose-response curve was fitted to the data to determine the toxicity (IC₅₀ value) of the drugs. The non-linear survival curves are presented as the mean ± SEM of four experiments performed in triplicate. Statistical significance was calculated using multiple <i>t</i>-test comparisons, where * p < 0.05 as compared with shNT. Inhibition of human shMRP2 expression reduced the resistance of Caco-2 cells to etoposide compared to control cells. IC₅₀ and RR values are presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0136080#pone.0136080.t001" target="_blank">Table 1</a>.</p

IC₅₀ and relative resistance (RR) values measured in response to etoposide, cisplatin and doxorubicin treatment of Caco-2 cells stably expressing non-target shRNA (shNT) or shMRP2.

<p>Caco-2 cells stably expressing shNT or shMRP2 (#305) were incubated with the cytotoxic drugs for 84 h. Sensitivity to the anti-cancer drug was determined by the MTT cell viability assay. Dose-response curves were fitted to the data to determine the toxicity (IC₅₀ value) of the drugs. Inhibition of human shMRP2 expression reduced the resistance of Caco-2 cells to etoposide, cisplatin and doxorubicin compared to control cells.</p><p>IC₅₀ and relative resistance (RR) values measured in response to etoposide, cisplatin and doxorubicin treatment of Caco-2 cells stably expressing non-target shRNA (shNT) or shMRP2.</p

ATP-dependent stimulation of MRP2 protein expression is strongly reduced in the presence of the P2 receptor antagonist Suramin.

<p>Caco-2 cells were treated with 100 μM PPADS or Suramin 30 min prior to the addition of 100 μM ATP for 6 h. MRP2 expression was analyzed by Western blotting. ATP stimulated the expression of MRP2 as compared to non-stimulated (N-S) cells, whereas the addition of Suramin prior to the ATP stimulation strongly decreased MRP2 expression compared to ATP-stimulated cells in the presence of vehicle (DMSO (-)) only. The presented blot is typical of three separate sets of experiments.</p