Transcriptional regulation of the human prostacyclin receptor gene is dependent on Sp1, PU.1 and Oct-1 in megakaryocytes and endothelial cells

Abstract

Prostacyclin plays a central role in haemostasis, inflammation and nociception. However, the factors regulating expression of the prostacyclin receptor (IP) gene in humans, or in other species, have not been identified. Herein it was 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 the vascular endothelial EA.hy 926 cell lines. Using deletion and genetic reporter analyses, the essential core promoter, termed PrmIP, was localized to -1022 to -895 proximal to the transcription initiation site, while an upstream repressor region, localized to -1502 to -1271, was also identified. Bioinformatic analysis revealed evolutionary conserved Sp1, PU.1 and Oct-1 sites within the core PrmIP and disruption of those elements each led to substantial reductions in PrmIP-directed gene expression in both HEL and EA.hy 926 cells. Electrophoretic mobility shift assays (EMSAs) and supershift assays established that Sp1, PU.1 and Oct-1 can bind to elements within the core promoter in vitro while chromatin immunoprecipitiation (ChIP) 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 EMSA 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, ChIP analysis established that neither Fli1 nor Ets-1 bind 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.