title: 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.
abstract: 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.
authors.affiliation: Department of Anatomy and Cell Biology, Faculty of Medicine and Health Sciences, Université de Sherbrooke, Sherbrooke, QC, Canada.
authors.forename: Djordje M
authors.initials: DM
authors.lastname: Grbic
authors.forename: Émilie
authors.initials: É
authors.lastname: Degagné
authors.forename: Jean-François
authors.initials: JF
authors.lastname: Larrivée
authors.forename: Maude S
authors.initials: MS
authors.lastname: Bilodeau
authors.forename: Valérie
authors.initials: V
authors.lastname: Vinette
authors.forename: Guillaume
authors.initials: G
authors.lastname: Arguin
authors.forename: Jana
authors.initials: J
authors.lastname: Stankova
authors.forename: Fernand-Pierre
authors.initials: FP
authors.lastname: Gendron
mesh_terms: D000818:Animals; D015153:Blotting, Western; D002478:Cells, Cultured; D054360:Chemokine CXCL1 / Q000235:genetics / Q000378:metabolism; D047369:Chromatin Immunoprecipitation; D004847:Epithelial Cells / Q000276:immunology* / Q000378:metabolism / Q000473:pathology; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D007249:Inflammation / Q000276:immunology* / Q000378:metabolism / Q000473:pathology; D015212:Inflammatory Bowel Diseases / Q000276:immunology* / Q000378:metabolism / Q000473:pathology; D016209:Interleukin-8 / Q000235:genetics* / Q000378:metabolism; D007413:Intestinal Mucosa / Q000276:immunology* / Q000378:metabolism / Q000473:pathology; D008156:Luciferases / Q000378:metabolism; D051379:Mice; D019950:Mitogen-Activated Protein Kinase 1 / Q000235:genetics / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000235:genetics / Q000378:metabolism; D020556:Neutrophil Infiltration; D010766:Phosphorylation; D012333:RNA, Messenger / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D018048:Receptors, Purinergic P2 / Q000235:genetics / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction; D018808:Transcription Factor AP-1 / Q000235:genetics / Q000378:metabolism*
chemical_list: D054360:Chemokine CXCL1; C475895:Cxcl1 protein, mouse; D016209:Interleukin-8; D012333:RNA, Messenger; D018048:Receptors, Purinergic P2; D018808:Transcription Factor AP-1; C099845:purinoceptor P2Y6; D008156:Luciferases; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3