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P2X7 RECEPTOR SIGNALLING IN THE DIFFERENTIATION OF INTESTINAL EPITHELIAL CELLS
K L’Ériger, FP Gendron
CIHR Team on the Digestive Epithelium, Département d’anatomie et de biologie cellulaire, FMSS, Université de Sherbrooke
AIMS: The ATP-gated P2X7 receptor channel (P2X7R) has long been associated to cell death. However, this particular receptor has recently been linked to other physiological functions, such as the modulation of the immune response and cell differentiation. Hypothesis: Activation of the P2X7R leads to the inhibition of intestinal epithelial cells (IECs) proliferation and participates to the differentiation process.
METHODS: Determination of P2X7R expression profile was determined by indirect immunofluorescence and western immunoblotting. Immunofluorescence localization of the P2X7R was performed on paraffin-embedded section of normal mouse jejunum and western blot on lysate of proliferative and differentiating Caco-2/15 cells. The effects of P2X7R activation on epithelial cells fate were determined by stimulating the cells with 1mM ATP or 100µM BzATP in a time dependent manner. The human P2X7R proximal promoter region was cloned by PCR in the pGL4.10-Luc expression vector (pGL4/P2X7). The possible interactions between transcription factors, involved in cell differentiation, and the promoter region of the P2X7R were determined by luciferase assays. Finally, modulation in expression and/or phosphorylation of key cell cycle proteins was monitored by western blot.
RESULTS: P2X7R expression was increased in differentiating Caco-2/15 cells. These results were correlated by indirect immunofluorescence studies where we observed an increase in P2X7R in the superior portion of mouse jejunum villus. Hence, luciferase assays reveals that P2X7R could be regulated at the transcriptional level by C/EBPalpha, C/EBPbeta and HNF-4alpha, all transcription factors known to be involved in cell differentiation. Finally, stimulation of IECs with ATP and BzATP resulted in an increase in p21Cip expression, as well as enhanced phosphorylation of PKCµ/PKD.
CONCLUSION: The increase in P2X7R expression along the crypt to villus axis of mouse jejunum and in differentiating Caco-2/15 cells as well as the transcriptional effect of C/EBPalpha, C/EBPbeta and HNF-4alpha suggest that P2X7R expression is regulated during IECs differentiation. While the inhibition of cell cycle progression modulated by the activation of the P2X7R suggests that this receptor could be a part of the mechanisms regulating the final differentiation of enterocytes.
This work was supported by NSERC and a FRSQ scholarship to FP Gendron