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 P2Y6 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 P2Y6 to CXCL8 expression in IEC.
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.
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 P2Y6 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.
This study not only describes the P2Y6 signaling mechanism regulating CXCL8 expression in IEC, but it also illustrates the potential of targeting P2Y6 to reduce intestinal inflammation. (Inflamm Bowel Dis 2012)
1 Department of Anatomy and Cell Biology, Faculty of Medicine and Health Sciences, Université de Sherbrooke, Sherbrooke, QC, Canada
2 Immunology Division, Department of Pediatrics, Faculty of Medicine and Health Sciences, Université de Sherbrooke, Sherbrooke, QC, Canada
3 Canadian Institutes of Health Research Team on Digestive Epithelium, Faculty of Medicine and Health Sciences, Université de Sherbrooke, Sherbrooke, QC, Canada
Reprints: Fernand-Pierre Gendron, PhD, Département d'anatomie et de biologie cellulaire, Faculté de médecine et des sciences de la santé, Université de Sherbrooke, 3001 12th Ave. North, Sherbrooke, QC, Canada, J1H 5N4
Received 26 September 2011; Accepted 29 September 2011
Published online 17 November 2011 in Wiley Online Library (wileyonlinelibrary.com).
Grant sponsor: Crohn's and Colitis Foundation of Canada; Grant Number: 2009-2012; Grant sponsor: Natural Sciences and Engineering Research Council of Canada; Grant Number: NSERC# 327128-06.