Epithelial cells participate in the immune response of the intestinal mucosa. Extracellular nucleotides have been recognized as inflammatory molecules. We investigated the role of extracellular nucleotides and their associated P2Y receptors in the secretion of cytokines by epithelial cells. The effect of intestinal inflammation on P2Y6 receptor expression was determined by PCR in the mouse, rat, and human. Localization of the P2Y6 receptor was determined by immunofluorescence microscopy in the colon of normal and dextran sulfate sodium-treated mice. The effect of P2Y6 activation by UDP on cytokine expression and release by epithelial cells was determined using a combination of Western blots, luciferase assays, RT-PCR, cytokine Ab arrays, and ELISA. Inflammation up-regulates P2Y2 as well as P2Y6 receptor expression in the mucosa of the colon of colitic mice. In vitro, we demonstrated that UDP could be released by Caco-2/15 cells. We have confirmed the increased expression of P2Y6 by challenging intestinal epithelial cell-6 and Caco-2/15 cells with TNF-α and IFN-γ and showing that stimulation of epithelial cells by UDP results in an increased expression and release of CXCL8 by an ERK1/2-dependent mechanism. The increase in CXCL8 expression was associated with a transcriptional activation by the P2Y6 receptor. This study is the first report demonstrating the implication of P2Y receptors in the inflammatory response of intestinal epithelial cells. We show for the first time that P2Y6, as well as P2Y2, expression is increased by the stress associated with intestinal inflammation. These results demonstrate the emergence of extracellular nucleotide signaling in the orchestration of intestinal inflammation.

BackgroundHnf4α, 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 Hnf4α in the maintenance of gut inflammatory homeostasis in mice.Methodology/Principal FindingsWe show here that specific epithelial deletion of Hnf4α 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 Hnf4α 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 Hnf4α 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 Hnf4α gene target in the intestinal epithelial context and is down-modulated in mouse experimental colitis and inflammatory bowel disease.ConclusionOur results highlight the critical role of Hnf4α to maintain intestinal inflammatory homeostasis during mouse adult life and uncover a novel function for Hnf4α in the regulation of claudin-15 expression. This establishes Hnf4α as a mediator of ion epithelial transport, an important process for the maintenance of gut inflammatory homeostasis.

Inflammatory stresses associated with inflammatory bowel diseases up-regulate P2Y2 mRNA receptor expression in the human colon adenocarcinoma cell line Caco-2, the noncancerous IEC-6 cells and in colonic tissues of patient suffering from Crohn’s disease and ulcerative colitis. However, the transcriptional events regulating P2Y2 receptor (P2Y2R) expression are not known. We have identified a putative transcription start site in the P2Y2R gene and demonstrated acetylation of Lys14 on histone H3 and Lys8 on histone H4, thus suggesting that the chromatin associated with the P2Y2 promoter is accessible to transcription factors. We also showed that the transcription factor NF-κB p65 regulates P2Y2R transcription under both proinflammatory and basal conditions. A NF-κB-responsive element was identified at −181 to −172 bp in the promoter region of P2Y2. Hence, activation of P2Y2R by ATP and UTP stimulated cyclooxygenase-2 expression and PGE2 secretion by intestinal epithelial cells. These findings demonstrate that P2Y2R expression is regulated during intestinal inflammation through an NF-κB p65-dependent mechanism and could contribute not only to inflammatory bowel disease but also to other inflammatory diseases by regulating PG release.

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