title: Regulation of the human prostacyclin receptor gene in megakaryocytes: major roles for C/EBPδ and PU.1.
abstract: The prostanoid prostacyclin plays a central role in haemostasis and vascular repair. Recent studies investigating the regulation of the human prostacyclin receptor (hIP) gene identified an upstream repressor region (URR) within its regulatory promoter, herein termed the PrmIP. This study aimed to identify the main trans-acting factors that bind within the URR to transcriptionally repress PrmIP-directed gene expression in the megakaryoblastic human erythroleukemia (HEL) 92.1.7 cell line. Of the putative cis-acting elements examined, disruption of C/EBP and PU.1 elements within the URR substantially increased PrmIP-directed gene expression. Chromatin immunoprecipitation (ChIP) confirmed that C/EBPδ and PU.1, but not C/EBPβ, bind to the URR in vivo, while ectopic expression of C/EBPδ substantially reduced hIP mRNA levels and PrmIP-directed gene expression. Phorbol 12-myristate 13-acetate (PMA)-induced megakaryocytic differentiation increased hIP mRNA and PrmIP-directed reporter gene expression and hIP-mediated cAMP generation in HEL cells. Two PMA-responsive regions, termed PRR1 and PRR2, were identified within PrmIP. Disruption of C/EBPδ and PU.1 cis-elements within the overlapping PRR1/URR and of Sp1, PU.1 and Oct-1 cis-elements within the overlapping PRR2/core PrmIP, revealed that both PRR1 and PRR2 contribute to the PMA- induction of hIP mRNA and gene expression in HEL cells. Furthermore, ChIP analysis established that induction of PrmIP-directed gene expression during megakaryocytic differentiation is largely regulated by PMA-induced dissociation of C/EBPδ and enhanced binding of PU.1 to PRR1 in addition to increased binding of Sp1, PU.1 and Oct-1 to elements within the core promoter/PRR2 in vivo. Taken together, these data provide critical insights into the transcriptional regulation of the hIP gene within the vasculature, including during megakaryocytic differentiation.
authors.affiliation: UCD Conway Institute of Biomolecular and Biomedical Research, University College, Dublin, Ireland.
authors.forename: Garret L
authors.initials: GL
authors.lastname: Keating
authors.forename: Elizebeth C
authors.initials: EC
authors.lastname: Turner
authors.forename: B Therese
authors.initials: BT
authors.lastname: Kinsella
mesh_terms: D002318:Cardiovascular Diseases / Q000378:metabolism; D015815:Cell Adhesion Molecules / Q000235:genetics / Q000378:metabolism; D002454:Cell Differentiation; D045744:Cell Line, Tumor; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D004915:Leukemia, Erythroblastic, Acute / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D008533:Megakaryocytes / Q000166:cytology / Q000378:metabolism* / Q000473:pathology; D000075983:Nectins; D050811:Octamer Transcription Factor-1 / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism; D044006:Receptors, Epoprostenol; D011982:Receptors, Prostaglandin / Q000235:genetics / Q000378:metabolism; D020218:Response Elements / Q000187:drug effects / Q000235:genetics*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism; D000099232:Proto-Oncogene Protein Spi-1
chemical_list: D015815:Cell Adhesion Molecules; D000075983:Nectins; D050811:Octamer Transcription Factor-1; D011518:Proto-Oncogene Proteins; D044006:Receptors, Epoprostenol; D011982:Receptors, Prostaglandin; D013755:Tetradecanoylphorbol Acetate; D015534:Trans-Activators; D000099232:Proto-Oncogene Protein Spi-1; C113936:PTGIR protein, human