Transcriptional Regulation of the Human Prostacyclin Receptor Gene Is Dependent on Sp1, PU.1 and Oct-1 in Megakaryocytes and Endothelial Cells

Prostacyclin plays a central role in hemostasis, inflammation and nociception. However, the factors reg-Ldating expression of the prostacyclin receptor (IP) gene in humans and in other species have not been identified. In this study, we sought to identify the key trans-acting factors and cis-acting elements regulating IP expression in the megakaryoblastic human erythroleukemia (HEL) 92.1.7 and vascular endothelial EA.hy 926 cell lines. With the use of deletion and genetic reporter analyses, the essential core promoter, termed PrmIP, was localized to the -1022 to -895 region proximal to the transcription initiation site, while an upstream repressor region, localized to -1502 to -1271, was also identified. Bioinformatic analysis revealed evolutionarily conserved Sp1, PU.1 and Oct-1 sites within the core PrmIP, and disruption of these elements each led to substantial reductions in PrmIP-directed gene expression in both HEL and EA.hy 926 cells. Electrophoretic mobility shift and supershift assays established that Sp1., PU.1 and Oct-1 can bind to elements within the core promoter in vitro, while chromatin immunoprecipitation assays confirmed their specific binding to chromatin in vivo. Furthermore, combination mutations of the Sp1, PU.1 and Oct-1. elements revealed that they act independently to co-regulate basal transcription of the IP gene, while ectopic expression of each of the trans-acting factors led to substantial increases in PrmIP-directed gene expression and IP mRNA expression in both HEL and EA.hy 926 cells. While electrophoretic mobility shift and antibody supershift assays established that the Ets family member Fli1, but not Ets-1, is capable of binding to the PU.1 element within PrmIP in vitro, chromatin immunoprecipitation analysis established that neither Fli1 nor Ets-1 binds to that element in vivo. Collectively, these data provide critical insights into the transcriptional regulation of the IP gene in human megakaryocytic and endothelial cells, identifying Sp1, PU.1 and Oct-1 as the critical factors involved in its basal regulation in humans. (c) 2008 Elsevier Ltd. All rights reserved.