C/EBPß regulates P2X7 receptor expression in response to glucose challenge in intestinal epithelial cells.

Activation of the ATP-dependent P2X7 receptor modulates glucose transport in intestinal epithelial cells through the downregulation of glucose transporter GLUT2. In the present study, we show that an increase in glucose concentration stimulates P2X7 receptor transcription via modulation of CCAAT/enhancer binding proteins (C/EBPs) α and ß expression. The described human P2X7 receptor promoter region (GenBank Y12851) was cloned upstream of a luciferase reporter gene in pGL4.10 plasmid and used to determine whether C/EBPs, namely C/EBPα and C/EBPß, are able to stimulate the transcription of P2X7 receptor. Results show that C/EBPß was the main regulator of P2X7 receptor expression in response to a glucose challenge. Chromatin immunoprecipitation (ChIP) assays further revealed that C/EBPß occupied the -213 to +6 nt P2X7 promoter region. Surprisingly, C/EBPα was also able to bind this region as revealed by ChIP assays, but without inducing receptor transcription. In fact, C/EBPα and the C/EBPß-LIP isoform blocked the C/EBPß-dependent regulation of P2X7 receptor transcription. These findings suggest that glucose is not only the major source of energy for cell function but may also act as a signaling molecule to stimulate the expression of regulatory proteins.

Glucose transporter 2 expression is down regulated following P2X7 activation in enterocytes.

With the diabetes epidemic affecting the world population, there is an increasing demand for means to regulate glycemia. Dietary glucose is first absorbed by the intestine before entering the blood stream. Thus, the regulation of glucose absorption by intestinal epithelial cells (IECs) could represent a way to regulate glycemia. Among the molecules involved in glycemia homeostasis, extracellular ATP, a paracrine signaling molecule, was reported to induce insulin secretion from pancreatic ß cells by activating P2Y and P2X receptors. In rat's jejunum, P2X7 expression was previously immunolocalized to the apex of villi, where it has been suspected to play a role in apoptosis. However, using an antibody recognizing the receptor extracellular domain and thus most of the P2X7 isoforms, we showed that expression of this receptor is apparent in the top two-thirds of villi. These data suggest a different role for this receptor in IECs. Using the non-cancerous IEC-6 cells and differentiated Caco-2 cells, glucose transport was reduced by more than 30% following P2X7 stimulation. This effect on glucose transport was not due to P2X7-induced cell apoptosis, but rather was the consequence of glucose transporter 2 (Glut2)'s internalization. The signaling pathway leading to P2X7-dependent Glut2 internalization involved the calcium-independent activation of phospholipase Cγ1 (PLCγ1), PKCδ, and PKD1. Although the complete mechanism regulating Glut2 internalization following P2X7 activation is not fully understood, modulation of P2X7 receptor activation could represent an interesting approach to regulate intestinal glucose absorption.

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.