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THE INCREASE IN P2Y2 NUCLEOTIDE RECEPTOR EXPRESSION IS REGULATED AT THE TRANSCRIPTIONAL LEVEL DURING INTESTINAL INFLAMMATION
E Degagné, D Grbic, FP Gendron
CIHR Team on the Digestive Epithelium, Département d’anatomie et de biologie cellulaire, FMSS, Université de Sherbrooke
AIM: Intestinal inflammation results in the upregulation of the P2Y2 nucleotide receptor (P2Y2R) expression in the colon of both DSS treated mice and in patients having Crohn disease and ulcerative colitis. However, the exact molecular mechanisms regulating the expression of the P2Y2R are not known. Hypothesis: Under inflammatory conditions, the transcriptional regulation of the P2Y2R is controlled by transcription factors C/EBPs and NFkappaB/p65.
METHODS: Proximal region of the human P2Y2R promoter was cloned into a pGL4.10-Luc vector (pGL4/P2Y2). Caco-2/15 cells were co-transfected with the pGL4/P2Y2 construction or with deletion mutants generated by restriction enzyme digestions and with the transcription factors C/EBPs or NFkappaB/p65. The interactions were determined by luciferase assays and identification of C/EBPs and NFkappaB/p65 potential binding sites determined by ChIP assays. Colitis was induced in mice with 5%DSS in water for 7 days. The colonic tissues were extracted and PCR assays were performed to evaluate the presence of P2Y2R. Tissue samples were also parafilm-embedded and immunofluorescence was performed for the detection of P2Y2R, C/EBPs and NFkappaB expression pattern.
RESULTS: We demonstrated that P2Y2R expression was up regulated in colitic mice. We established that the transcription factors C/EBPs and NFkappaB/p65 can bind to the promoter region of P2Y2 and transactivated the luciferase gene. We also demonstrated, in vivo by ChIP assays, that these transcription factors can bind to specific region of the P2Y2R proximal promoter.
CONCLUSION: These results showed that inflammation up-regulates the expression of the P2Y2R and that the expression of this gene is regulated at the transcriptional level by C/EBPs and NFkappaB.
This work was supported by the CCFC and the FRSQ scholarship program to FP Gendron.