P2Y2 receptor expression is regulated by C/EBPβ during inflammation in intestinal epithelial cells

Inflammatory bowel diseases are characterized by relapses and remission periods during which numerous factors, including stress factors and nucleotides, are mobilized to re-establish intestinal mucosal homeostasis. We have previously found that expression of the P2Y2 nucleotide receptor is increased in colonic tissue isolated from inflammatory bowel disease patients as well as in a mouse model of colitis, and that P2Y2 transcription is regulated in part by nuclear factor ?B (NF-?B) p65. Transcription factor DNA-binding site analysis identified three potential CCAAT/enhancer-binding protein beta (C/EBP beta) binding sites in the P2Y2 proximal promoter. We then assessed the role of C/EBP transcription factors in the regulation of P2Y2 in intestinal epithelial cells (IECs). We identified a region between -229 and -220 bp upstream of the transcription initiation site as a DNA-binding site for C/EBP beta, by electrophoretic mobility and supershift assays. Mutagenesis of this site decreased C/EBP beta-dependent P2Y2 expression, as assessed by luciferase assays. In vivo, C/EBP beta as well as P2Y2 expression was increased in colonic IECs isolated from mice with dextran sulfate sodium-induced acute colitis. In contrast, P2Y2 expression was decreased in C/EBP beta-deficient mice treated with dextran sulfate sodium. Although C/EBP beta was sufficient to induce P2Y2 transcription, the effect of C/EBP beta and NF-?B p65 on receptor transcription was synergistic. Chromatin immunoprecipitation assays revealed that both proteins simultaneously bind to the P2Y2 promoter. Thus, we have identified C/EBP beta as a novel regulator of P2Y2 expression.