P2Y2 receptor promotes intestinal microtubule stabilization and mucosal re-epithelization in experimental colitis.

P2Y(2) receptor expression is increased in intestinal epithelial cells (IECs) during inflammatory bowel diseases (IBDs). In this context, P2Y(2) stimulates PGE(2) release by IECs, suggesting a role in wound healing. For this study, we have used the non-cancerous IEC-6 cell line. IEC-6 cell migration was determined using Boyden chambers and the single-edged razor blade model of wounding. The receptor was activated using ATP, UTP, or 2-thioUTP. Pharmacological inhibitors, a blocking peptide, a neutralizing antibody and interfering RNAs were used to characterize the signaling events. Focal adhesions and microtubule (MT) dynamics were determined by immunofluorescence using anti-vinculin and anti-acetylated-α-tubulin antibodies, respectively. In vivo, the dextran sodium sulfate mouse model of colitis was used to characterize the effects of P2Y(2) agonist 2-thioUTP on remission. We showed that P2Y(2) increased cell migration and wound closure by recruiting Go protein with the cooperation of integrin α(v) . Following P2Y(2) activation, we demonstrated that GSK3ß activity was inhibited in response to Akt activation. This leads to MT stabilization and increased number of focal adhesions. In vivo, P2Y(2) activation stimulates remission, as illustrated by a reduction in the disease activity index values and histological scores as compared to control mice. These findings highlight a novel function for this receptor in IECs. They also illustrate that P2Y receptors could be targeted for the development of innovative therapies for the treatment of IBDs.

P2Y2 receptor transcription is increased by NF-kappa B and stimulates cyclooxygenase-2 expression and PGE2 released by intestinal epithelial cells.

Inflammatory stresses associated with inflammatory bowel diseases up-regulate P2Y(2) 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 P2Y(2) receptor (P2Y(2)R) expression are not known. We have identified a putative transcription start site in the P2Y(2)R gene and demonstrated acetylation of Lys(14) on histone H3 and Lys(8) on histone H4, thus suggesting that the chromatin associated with the P2Y(2) promoter is accessible to transcription factors. We also showed that the transcription factor NF-kappaB p65 regulates P2Y(2)R transcription under both proinflammatory and basal conditions. A NF-kappaB-responsive element was identified at -181 to -172 bp in the promoter region of P2Y(2). Hence, activation of P2Y(2)R by ATP and UTP stimulated cyclooxygenase-2 expression and PGE(2) secretion by intestinal epithelial cells. These findings demonstrate that P2Y(2)R expression is regulated during intestinal inflammation through an NF-kappaB p65-dependent mechanism and could contribute not only to inflammatory bowel disease but also to other inflammatory diseases by regulating PG release.

P2Y6 receptor contributes to neutrophil recruitment to inflamed intestinal mucosa by increasing CXC chemokine ligand 8 expression in an AP-1-dependent manner in epithelial cells.

BACKGROUND: Inflammatory bowel diseases are characterized by the presence of CXCL8 at the site of lesions resulting in neutrophil recruitment and loss of tissue functions. We report that P2Y(6) receptor activation stimulates CXCL8 expression and release by intestinal epithelial cells (IECs). In this context, we investigated if uridine 5'-diphosphate (UDP) enemas stimulate neutrophil recruitment to the mucosa of mice suffering from colitis-like disease and we characterized the signaling events linking P2Y(6) to CXCL8 expression in IEC. METHODS: Neutrophil recruitment was monitored by immunofluorescence and FACS analysis. Expression of Cxcl1, a mouse functional homolog of CXCL8, was determined by quantitative real-time polymerase chain reaction (qPCR). Pharmacological inhibitors and interfering RNAs were used to characterize the signaling pathway. The outcomes of these treatments on protein phosphorylation and on CXCL8 expression were characterized by western blots, qPCR, luciferase, and chromatin immunoprecipitation (ChIP) assays. RESULTS: Mutation of the AP-1 site in the CXCL8 core promoter abolished the UDP-stimulating effect. The c-fos/c-jun dimer was identified as the AP-1 complex regulating CXCL8 in response to UDP stimulation. Regulation of CXCL8 expression by P2Y(6) required PKCδ activation upstream of the signaling pathway composed of MEK1/2-ERK1/2 and c-fos. UDP administration to mice suffering from colitis-like disease increased the number of neutrophil infiltrating the mucosa, correlating with Cxcl1 increased expression in IEC and the severity of inflammation. CONCLUSIONS: This study not only describes the P2Y(6) signaling mechanism regulating CXCL8 expression in IEC, but it also illustrates the potential of targeting P2Y(6) to reduce intestinal inflammation.

Intestinal inflammation increases the expression of the P2Y6 receptor on epithelial cells and the release of CXC chemokine ligand 8 by UDP.

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 P2Y(6) receptor expression was determined by PCR in the mouse, rat, and human. Localization of the P2Y(6) receptor was determined by immunofluorescence microscopy in the colon of normal and dextran sulfate sodium-treated mice. The effect of P2Y(6) 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 P2Y(2) as well as P2Y(6) 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 P2Y(6) by challenging intestinal epithelial cell-6 and Caco-2/15 cells with TNF-alpha and IFN-gamma 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 P2Y(6) 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 P2Y(6), as well as P2Y(2), 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.